Back to EveryPatent.com
United States Patent |
6,261,802
|
Huang
,   et al.
|
July 17, 2001
|
Ups (ugc)
Abstract
The invention provides ups (ugc) polypeptides and polynucleotides encoding
ups (ugc) polypeptides and methods for producing such polypeptides by
recombinant techniques. Also provided are methods for utilizing ups (ugc)
polypeptides to screen for antibacterial compounds.
Inventors:
|
Huang; Jianzhong (Schwenksville, PA);
Jiang; Xinhe (Royersford, PA);
McDevitt; Damien (Berwyn, PA);
Traini; Christopher M. (Media, PA)
|
Assignee:
|
SmithKline Beecham Corporation (Philadelphia, PA)
|
Appl. No.:
|
175014 |
Filed:
|
October 19, 1998 |
Current U.S. Class: |
435/69.1; 435/6; 435/253.3; 435/320.1; 435/325; 536/23.7 |
Intern'l Class: |
C12P 021/06; C12N 015/00 |
Field of Search: |
536/23.7
435/69.1,6,69.7,71.1,320.1,253.3,325
|
References Cited
Other References
Taguchi et al., Gene, 172(1):165-166, 1996.*
Shimizu, et al., "Molecular Cloning, Expression, and Purification of
Undecaprenyl Diphosphate Synthase", The Journal of Biological Chemistry,
vol. 273, No. 31, pp. 19476-19481, Jul. 31, 1998.
|
Primary Examiner: Allen; Marianne P.
Attorney, Agent or Firm: Gimmi; Edward R., Diebert; Thomas S., King; William T.
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of U.S. patent application Ser.
No. 08/992,421 which was filed Dec. 17, 1997 now abandoned.
Claims
What is claimed is:
1. An isolated polynucleotide segment comprising a nucleic acid sequence
that encodes a polypeptide comprising the amino acid sequence of SEQ ID
NO:2, wherein the nucleic acid sequence is not genomic DNA.
2. A vector comprising the isolated polynucleotide segment of claim 1.
3. An isolated host cell comprising the vector of claim 2.
4. A process for producing a polypeptide comprising culturing the host cell
of claim 3 under conditions sufficient for the production of the
polypeptide, wherein the polypeptide comprises SEQ ID NO:2.
5. An isolated polynucelotide segment comprising the full complement of the
nucleotide sequence of claim 1, wherein the full complement is not genomic
DNA and wherein the full complement detects Pseudomonas aeruginosa by
hybridization.
6. An isolated polynucleotide segment comprising SEQ ID NO:1, wherein the
polynucleotide segment is not genomic DNA.
7. A vector comprising the isolated polynucleotide segment of claim 6.
8. An isolated host cell comprising the vector of claim 7.
9. A process for producing a polypeptide comprising culturing the host cell
of claim 8 under conditions sufficient for the production of the
polypeptide, wherein the polypeptide comprises SEQ ID NO:2.
10. An isolated polynucleotide segment comprising the full complement of
the nucleotide sequence of claim 6, wherein the full complement is not
genomic DNA and wherein the full complement detects Pseudomonas aeruginosa
by hybridization.
11. An isolated polynucleotide comprising a nucleic acid sequence which
encodes a polypeptide consisting of the amino acid sequence of SEQ ID
NO:2, wherein the nucleic acid sequence is not genomic DNA.
12. A vector comprising the isolated polynucleotide of claim 11.
13. An isolated host cell comprising the vector of claim 12.
14. A process for producing a polypeptide comprising culturing the host
cell of claim 13 under conditions sufficient for the production of the
polypeptide, wherein the polypeptide consists of SEQ ID NO:2.
Description
FIELD OF THE INVENTION
This invention relates to newly identified polynucleotides and
polypeptides, and their production and uses, as well as their variants,
agonists and antagonists, and their uses. In particular, the invention
relates to polynucleotides and polypeptides of the ups (undecaprenyl
diphosphate synthase) family, as well as their variants, hereinafter
referred to as "ups(ugc)," "(ugc) polynucleotide(s)," and "ups (ugc)
polypeptide(s)" as the case may be.
BACKGROUND OF THE INVENTION
The frequency of Pseudomonas aeruginosa infections has risen dramatically
in the past few decades. This has been attributed to the emergence of
multiply antibiotic resistant strains and an increasing population of
people with weakened immune systems. It is no longer uncommon to isolate
Pseudomonas aeruginosa strains which are resistant to some or all of the
standard antibiotics. This phenomenon has created an unmet medical need
and demand for new anti-microbial agents, vaccines, drug screening
methods, and diagnostic tests for this organism.
Moreover, the drug discovery process is currently under going a fundamental
revolution as it embraces "functional genomics," that is, high throughput
genome- or gene-based biology. This approach is rapidly superseding
earlier approaches based on "positional cloning" and other methods.
Functional genomics relies heavily on the various tools of bioinformatics
to identify gene sequences of potential interest from the many molecular
biology databases now available as well as from other sources. There is a
continuing and significant need to identify and characterize further genes
and other polynucleotides sequences and their related polypeptides, as
targets for drug discovery.
Clearly, there exists a need for polynucleotides and polypeptides, such as
the ups (ugc) embodiments of the invention, that have a present benefit
of, among other things, being useful to screen compounds for antimicrobial
activity. Such factors are also useful to determine their role in
pathogenesis of infection, dysfunction and disease. There is also a need
for identification and characterization of such factors and their
antagonists and agonists to find ways to prevent, ameliorate or correct
such infection, dysfunction and disease.
SUMMARY OF THE INVENTION
The present invention relates to ups (ugc), in particular ups (ugc)
polypeptides and ups (ugc) polynucleotides, recombinant materials and
methods for their production. In another aspect, the invention relates to
methods for using such polypeptides and polynucleotides, including
treatment of microbial diseases, amongst others. In a further aspect, the
invention relates to methods for identifying agonists and antagonists
using the materials provided by the invention, and for treating microbial
infections and conditions associated with such infections with the
identified agonist or antagonist compounds. In a still further aspect, the
invention relates to diagnostic assays for detecting diseases associated
with microbial infections and conditions associated with such infections,
such as assays for detecting ups (ugc) expression or activity.
Various changes and modifications within the spirit and scope of the
disclosed invention will become readily apparent to those skilled in the
art from reading the following descriptions and from reading the other
parts of the present disclosure.
DESCRIPTION OF THE INVENTION
The invention relates to ups (ugc) polypeptides and polynucleotides as
described in greater detail below. In particular, the invention relates to
polypeptides and polynucleotides of a ups (ugc) of Pseudomonas aeruginosa
which is related by amino acid sequence homology to Micrococcus luteus Ups
(ugc) polypeptide. The invention relates especially to ups (ugc) having
the nucleotide and amino acid sequences set out in Table 1 as SEQ ID
NO:1and SEQ. ID NO:2 respectively. Note that sequences recited in the
Sequence Listing below as "DNA" represents an exemplification of the
invention, since those of ordinary skill will recognize that such
sequences can be usefully employed in polynucleotides in general,
including ribopolynucleotides.
TABLE 1
Ups (ugc) Polynucleotide and Polypeptide Sequences
(A) Pseudomonas aeruginosa ups (ugc) polynecleotide sequence
[SEQ ID NO:1].
5'-ATGGAAAAGACCCGGAAGGATGTGTGCGTGCCACGCCACGTGGCCATTATCATGGACGGT
AACAATCGCTGGGCGAAGAAGCGTCTTCTGCCCGGCGTCGCCGGCCACAAGGCCGGTGTC
GATGCCGTCAGGGCGGTGATCGAGGTCTGCGCCGAGGCAGGGGTCGAGGTCCTCACCCTG
TTCGCGTTCTCCAGCGAGAACTGGCAGCGTCCGGCGGACGAAGTCAGCGCGCTGATGGAG
CTGTTTCTCGTGGCCCTGCGCCGCGAGGTGCGCAAGCTCGACGAGAACGGCATCCGCCTG
CGCATCATCGGCGATCGCACGCGTTTCCATCCGGAGTTGCAGGCGGCCATGCGCGAAGCG
GAAGCCGCCACTGCCGGCAATACCCGTTTCCTCCTCCAGGTCGCCGCCAACTACGGCGGC
CAGTGGGACATCGTCCAGGCCGCACAGCGCCTGGCGCGCGAGGTCCAGGGCGGGCACCTG
GCGGCGGACGATATCTCCGCCGAGCTGCTCCAGGGCTGCCTGGTGACCGGCGACCAGCCG
CTGCCCGACCTGTGCATCCGCACCGGCGGCGAGCATCGCATCAGCAATTTCCTTCTCTGG
CAGCTGGCCTACGCCGAGCTGTATTTCTCCGACCTGTTCTGGCCCGACTTCAAGCACGCG
GCGATGCGGGCTGCCCTGGCGGATTTCTCCAAGCGCCAGCGCCGCTTCGGCAAGACCAGC
GAGCAAGTCGAGGCCGAAGCCCGTCCGTCATGCTGA
-3'
(B) Pseudomonas aeruginosa ups (ugc) polypeptide sequence deduced
from a polynucleotide sequence in this table [SEQ ID NO:2].
NH.sub.2 -MEKTRKDVCVPRHVAIIMDGNNRWAKKRLLPGVAGHKAGVDAVRAVIEVCAEAGVEVLTL
FAFSSENWQRPADEVSALMELFLVALRREVRKLDENGIRLRIIGDRTRFHPELQAAMREA
EAATAGNTRFLLQVAANYGGQWDIVQAAQRLAREVQGGHLAADDISAELLQGCLVTGDQP
LPDLCIRTGGEHRISNFLLWQLAYAELYFSDLFWPDFKHAAMRAALADFSKRQRRFGKTS
EQVEAEARPSC-COOH
Polypeptides
Ups (ugc) polypeptide of the invention is substantially phylogenetically
related to other proteins of the ups (undecaprenyl diphosphate synthase)
family.
In one aspect of the invention there are provided polypeptides of
Pseudomonas aeruginosa referred to herein as "ups (ugc)" and "ups (ugc)
polypeptides" as well as biologically, diagnostically, prophylactically,
clinically or therapeutically useful variants thereof, and compositions
comprising the same.
Among the particularly preferred embodiments of the invention are variants
of ups (ugc) polypeptide encoded by naturally occurring alleles of the ups
(ugc) gene.
The present invention further provides for an isolated polypeptide which:
(a) comprises of consists of and amino acid sequence which has at least
70% identity, preferable at least 80% identity, more preferably at least
90% identity, yet more preferably at least 95% identity, most preferably
at least 97-99% or exact identity, to that of SEQ ID NO:2 over the entire
length of SEQ ID NO:2; (b) a polypeptide encoded by an isolated
polynucleotide comprising or consisting of a polynucleotide sequence which
has at least 70% identity, preferably at least 80% identity, more
preferably at least 90% identity, yet more preferably at least 95%
identity, even more preferably at least 97-99% or exact identity to SEQ ID
NO:1 over the entire length of SEQ ID NO:1; (c) a polypeptide encoded by
an isolated polynucleotide comprising or consisting of polynucleotide
sequence encoding a polypeptide which has a least 70% identity, preferably
at least 80% identity, more preferably at least 90% identity, yet more
preferably at least 95% identity, even more preferably at least 97-99% or
exact identity, to the amino acid sequence of SEQ ID NO:2, over the entire
length of SEQ ID NO:2.
The polypeptides of the invention include a polypeptide of Table 1 [SEQ ID
NO:2] (in particular the mature polypeptide) as well as polypeptides and
fragments, particularly those which have the biological activity of ups
(ugc), and also those which have at least 70% identity to a polypeptide of
Table 1 [SEQ ID NO:1] or the relevant portion, preferably at least 89%
identity to a polypeptide of Table 1 [SEQ ID NO:2] and more preferably at
least 90% identity to a polypeptide of Table 1 [SEQ ID NO:2] and still
more preferably at least 95% identity to a polypeptide of Table 1 [SEQ ID
NO:2] and also include portions of such polypeptides with such portion of
the polypeptide generally containing at least 30 amino acids and more
preferably at least 50 amino acids.
the invention also includes a polypeptide consisting of or comprising a
polypeptide of the formula:
X--(R.sub.1).sub.m --(R.sub.2)--(R.sub.3).sub.n --Y
wherein, at the amino terminus, X is hydrogen, a metal or any other moiety
described herein for modified polypeptides, and at the carboxyl terminus,
Y is hydrogen, a metal or any other moiety described herein for modified
polypeptides, T.sub.1 and R.sub.3 are any amino acid residue or modified
amino acid residue, m is an integer between 1 and 1,000 or zero, n is an
integer between 1 and 1,000 or zero, and R.sub.2 is an amino acid sequence
of the invention, particularly an amino acid sequence selected from Table
1 or modified forms thereof. In the formula above, T.sub.2 is oriented so
that its amino terminal amino acid residue is at the left, covalently
bound to R.sub.1, and its carboxy terminal amino acid residue is at the
right, covalently bound to R.sub.3. Any stretch of amino acid residues
denoted by either R.sub.1 or R.sub.3, where m and/or n is greater than 1,
may be either a heteropolymer or a homopolymer, preferably a
heteropolymer. Other preferred embodiments of the invention are provided
where m is an integer between 1 and 50, 100 or 500, and n is an integer
between 1 and 50, 100, or 500.
It is most preferred that a polypeptide of the invention is derived from
Pseudomonas aeruginosa, however, it may preferably be obtained from other
organisms of the same taxonomic genus. A polypeptide of the invention may
also be obtained, for example, from organisms of the same taxonomic family
or order.
A fragment is a variant polypeptide having an amino acid sequence that is
entirely the same as part but not all of any amino acid sequence of any
polypeptide of the invention. As with ups (ugc) polypeptides, fragments
may be "free-standing," or comprised within a larger polypeptide of which
they form a part or region, most preferably as a single continuous region
in a single larger polypeptide.
Preferred fragments include, for example, truncation polypeptides having a
portion of an amino acid sequence of Table 1 [SEW ID NO:2], or of variants
thereof, such as a continuous series of residues that includes an amino-
and/or carboxyl-terminal amino acid sequence. Degradation forms of the
polypeptides of the invention produced by or in a host cell, particularly
a Pseudomonas aeruginosa, are also preferred. Further preferred are
fragments characterized by structural or functional attributes such as
fragments that comprise alpha-helix and alpha-helix forming regions,
beta-sheet and beta-sheet-forming regions, turn and turn-forming regions,
coil and coil-forming regions, hydrophilic regions, hydrophobic regions,
alpha amphipathic regions, beta amphipathic regions, flexible regions,
surface-forming regions, substrate binding region, and high antigenic
index regions.
Further preferred fragments include an isolated polypeptide comprising an
amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous
amino acids from the amino acid sequence of SEQ ID NO:2, or an isolated
polypeptide comprising an amino acid sequence having at least 15, 20, 30,
40, 50 or 100 contiguous amino acids truncated or deleted from the amino
acid sequence of SEQ ID NO:2.
Also preferred are biologically active fragments which are those fragments
that mediate activities of ups (ugc), including those with a similar
activity or an improved activity or with a decreased undesirable activity.
Also included are those fragments that are antigenic or immunogenic in an
animal, especially in a human. Particularly preferred are fragments
compassing receptors or domains of enzymes that confer a function
essential for viability of Pseudomonas aeruginosa or the ability to
initiate, or maintain cause Disease in an individual, particularly a
human.
Fragments of the polypeptides of the invention may be employed for
producing the corresponding full-length polypeptide by peptide synthesis;
therefore, these variants may be employed as intermediates for producing
the full-length polypeptides of the invention.
In addition to the standard single and triple letter representations for
amino acids, the term "X" or "Xaa" may also be used in describing certain
polypeptides of the invention. "X" and "Xaa"mean that any of the twenty
naturally occurring amino acids may appear at such a designated position
in the polypeptide sequence.
Polynucleotides
It is an object of the invention to provide polynucleotides that encode ups
(ugc) polypeptides, particularly polynucleotides that encode the
polypeptide herein designated ups (ugc).
In a particularly preferred embodiment of the invention the polynucleotide
comprises a region encoding ups (ugc) polypeptides comprising a sequence
set out in Table 1 [SEQ ID NO:1] which includes a full length gene, or a
variant thereof. The Applicants believe that this full length gene is
essential to the growth and/or survival of an organism which possesses it,
such as Pseudomonas aeruginosa.
As a further aspect of the invention there are provided isolated nucleic
acid molecules encoding and/or expressing ups (ugc) polypeptides and
polynucleotides, particularly Pseudomonas aeruginosa ups (ugc)
polypeptides and polynucleotides, including, for example, unprocessed RNAs
ribozyme RNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs. Further
embodiments of the invention include biologically, diagnostically,
prophylactic ally, clinically or therapeutically useful polynucleotides
and polypeptides, and variants thereof, and compositions comprising the
same.
Another aspect of the invention relates to isolated polynucleotides,
including at least one full length gene, that encodes a ups (ugc)
polypeptide having a deduced amino acid sequence of Table 1 [SEQ ID NO:2],
and polynucleotides closely related thereto and variants thereof.
In another particularly preferred embodiment of the invention there is a
ups (ugc) polypeptide from Pseudomonas aeruginosa comprising or consisting
of an amino acid sequence of Table 1 [SEQ ID NO:2], or a variant thereof.
Using the information provided herein , such as a polynucleotide sequence
set out in Table 1 [SEQ ID NO:2], a polynucleotide of the invention
encoding ups (ugc) polypeptide may be obtained using standard cloning and
screening methods, such as those for cloning and sequencing chromosomal
DNA fragments from bacteria using [Pseudomonas aeruginosa Pseudomonas
aeruginosa strain 4 cells as starting material, followed by obtaining a
full length clone. For example, to obtain a polynucleotide sequence of the
invention, such as a polynucleotide sequence given in Table 1 [SEQ ID
NO:1], typically a library of clones of chromosomal DNA of Pseudomonas
aeruginosa Pseudomonas aeruginosa strain 4 in E. coli or some other
suitable host is probed with a radiolabeled oligonucleotide, preferably a
17-mer or longer, derived from a partial sequence. Clones carrying DNA
identical to that of the prove can then be distinguished using stringent
hybridization Conditions. By sequencing the individual clones thus
identified by hybridization with sequencing primers designed from the
original polypeptide or polynucleotide sequence it is then possible to
extend the polynucleotide sequence in both directions to determine a full
length gene sequence. Conveniently, such sequencing is performed, for
example, using denatured double stranded DNA prepared from a plasmid
clone. Suitable techniques are described by Maniatis, T., Fritsch, E. F.
and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.; Cold
spring Harbor Laboratory Press, cold spring Harbor, N.Y. (1989). (see in
particular Screening by Hybridization 1.90 and sequencing Denatured
double-Stranded DNA Templates 13.70). Direct genomic DNA sequencing may
also be performed to obtain a full length gene sequence. Illustrative of
the invention, each polynucleotide set out in Table 1 [SEQ ID NO:1] was
discovered in a DNA library derived from Pseudomonas aeruginosa
Pseudomonas aeruginosa strain 4.
Moreover, each DNA sequence set out in Table 1 [SEQ ID NO:1] contains an
open reading frame encoding a protein having about the number of amino
acid residues set forth in Table 1 [SEQ ID NO:2] with a deduced molecular
weight that can be calculated using amino acid residue molecular weight
values well known to those skilled in the art. The polynucleotide of SEQ
ID NO:1], encodes the polypeptide of SEQ ID NO:2.
In a further aspect, the present invention provides for an isolated
polynucleotide comprising or consisting of: (a) a polynucleotide sequence
which has at least 70% identity, preferably at least 0% identity, more
preferably at least 90% identity, yet more preferably at least 95%
identity, even more preferably at least 97-99% or exact identity to SEQ ID
NO:1 over the entire length of SEQ ID NO:1; (b) a polynucleotide sequence
encoding a polypeptide which has at least 70% identity, preferably at
least 80% identity, more preferably at least 90% identity, yet more
preferably at least 95% identity, even more preferably at least 97-99% or
100% exact, to the amino acid sequence of SEQ ID NO:2, over the entire
length of SEQ ID NO:2.
A polynucleotide encoding a polypeptide of the present invention, including
homologs and orthologs from species other than Pseudomonas aeruginosa, may
be obtained by process which comprises the steps of screening an
appropriate library under stringent hybridization conditions with a
labeled or detectable probe consisting of or comprising the sequence of
SEQ ID NO:1 or a fragment thereof; and isolating a full-length gene and/or
genomic clones containing said polynucleotide sequence.
The invention provides a polynucleotide sequence identical over its entire
length to a coding sequence (open reading frame) in Table 1 [SEQ ID NO:1].
Also provided by the invention is a coding sequence for a mature
polypeptide or a fragment thereof, by itself as well as a coding sequence
for a mature polypeptide or a fragment in reading frame with another
coding sequence, such as a sequence encoding a leader or secretory
sequence, a pre-, or pro- or prepro-protein sequence. The polynucleotide
of the invention may also contain at least one non-coding sequence,
including for example, but not limited to at least one non-coding 5' and
3' sequence, such as the transcribed but non-translated sequences,
termination signals (such as rho-dependent and rho-independent termination
signals), ribosome binding sites, Kozak sequences, sequences that
stabilize mTNA, introns, and polyadenylation signals. The polynucleotide
sequence may also comprise additional coding sequence encoding additional
amino acids. For example, a marker sequence that facilitates purification
of the fused polypeptide can be encoded. In certain embodiments of the
invention, the marker sequence is a hexa-histidine peptide, as provided in
the pQE vector (Qiagen, Inc.) and described in Gentz et ak,m Proc. Natl.
Acad. Sci., USA 86: 821-824 (1989), or an HA peptide tag (Wilson et al.,
Cell 37: 767 (1984), both of which may be useful in purifying polypeptide
sequence fused to them. Polynucleotides of the invention also include, but
are not limited to, polynucleotides comprising a structural gene and its
naturally associated sequences that control gene expression.
A preferred embodiment of the invention is a polynucleotide of consisting
of or comprising nucleotide 1 to the nucleotide immediately upstream of or
including nucleotide 754 set forth in SEQ ID NO:1 of Table 1, both of
which encode the ups (ugc) polypeptide.
The invention also includes a polynucleotide consisting of or comprising a
polynucleotide of the formula
X--(R.sub.1).sub.m --(R.sub.2)--(R.sub.3).sub.n --Y
wherein, the 5' end of the molecule, X is hydrogen, a metal or a modified
nucleotide residue, or together with Y defines a covalent bond, and at the
3' end of the molecule, Y is hydrogen, a metal, or a modified nucleotide
residue, or together with X defines the covalent bond, each occurrence of
R.sub.1 and R.sub.3 is independently any nucleic acid residue or modified
nucleic acid residue, m is an integer between 1 and 3000 or zero, n is an
integer between 1 and 3000 or zero, and R.sub.2 is a nucleic acid sequence
or modified nucleic acid sequence of the invention, particularly a nucleic
acid sequence selected from Table 1 or a modified nucleic acid sequence
thereof. In the polynucleotide formula above, R.sub.2 is oriented so that
its 5' end nucleic acid residue is a the left, bound to Rv1, and its 3'
end nucleic acid residue is at the right, bound to R.sub.3. Any stretch of
nucleic acid residues denoted by either .sub.2 and/or T.sub.2, where m
and/or n is greater than 1, may be either a heteropolymer or a
homopolymer, preferably a heteropolymer. Where, in a preferred embodiment,
X and Y together define a covalent bond, the polynucleotide of the above
formula is a closed, circular polynucleotide, which can be a
double-stranded polynucleotide wherein the formula shows a first strand to
which the second strand is complementary. In other preferred embodiment m
and/or n is and integer between 1 and 50, 100 or 500, and n is an integer
between 1 and 50, 100, and 500.
It is most preferred that a polynucleotide of the invention is derived from
Pseudomonas aeruginosa, however, it may preferably be obtained from other
organisms of the same taxonomic genus. A polynucleotide of the invention
may also be obtained, for example, from organisms of the same taxonomic
family or order.
The term "polynucleotide encoding a polypeptide" as used herein encompasses
polynucleotides that include a sequence encoding a polypeptide of the
invention, particularly a bacterial polypeptide and more particularly a
polypeptide of the Pseudomonas aeruginosa ups (ugc) having an amino acid
sequence set out in Table 1 [SEQ ID NO:2]. The term also encompasses
polynucleotides that include a single continuous region or discontinuous
regions encoding the polypeptide (for example, polynucleotides interrupted
by integrated phage, an integrated insertion sequence, and integrated
vector sequence, an integrated transposon sequence, or due to RNA editing
or genomic DNA reorganization) together with additional regions, that also
may contain coding and/or non-coding sequences.
The invention further relates to variants of the polynucleotides described
herein that encode variants of a polypeptide having a deduced amino acid
sequence of Table 1 [SEQ ID NO:2]. Fragments of a polynucleotides of the
invention may be used, for example, to synthesize full-length
polynucleotides of the invention.
Further particularly preferred embodiments are polynucleotides encoding ups
(ugc) variants, that have the amino acid sequence of ups (ugc) polypeptide
of Table 1 [SEQ ID NO:2] in which several, a few, 5 or 10, 1 to 3, 2, 1 or
no amino acid residues are substituted, modified, deleted an/or added, in
any combination. Especially preferred among these are silent
substitutions, additions and deletions, that do not alter the properties
and activities of ups (ugc) polypeptide.
Further preferred embodiments of the invention are polynucleotides that are
at least 7% identical over their entire length to a polynucleotide
encoding ups (ugc) polypeptide having an amino acid sequence set out in
Table 1 [SEQ ID NO:2], and polynucleotides that are complementary to such
polynucleotides. Alternatively, most highly preferred are polynucleotides
that comprise a region that is at least 80% identical over its entire
length to a polynucleotide encoding ups (ugc) polypeptide and
polynucleotides complementary thereto. In this regard, polynucleotides at
least 90% identical over their entire length to the same are particularly
preferred, and among these particularly preferred polynucleotides, those
with at least 95% are especially preferred. Furthermore, those with at
least 97% are highly preferred among those with at least 95%, and among
these those with at least 98% and at least 99% are particularly highly
preferred, with at least 99% being the more preferred.
Preferred embodiments are polynucleotides encoding polypeptides that retain
substantially the same biological function or activity as the mature
polypeptide encoded by a DBA of Table 1 [SEQ ID NO:1].
In accordance with certain preferred embodiments of this invention there
are provided polynucleotides that hybridize, particularly under stringent
conditions, to ups (ugc) polynucleotide sequences, such as those
polynucleotides in Table 1.
The invention further relates to polynucleotides that hybridize to the
polynucleotide sequences provided herein. In this regard, the invention
especially relates to polynucleotides that hybridize under stringent
conditions to the polynucleotides described herein. As herein used, the
terms "stringent conditions" and "stringent hybridization conditions" mean
hybridization occurring only if there is a least 95% and preferably at
least 97% identify between the sequences. A specific example of stringent
hybridization conditions is overnight incubation at 42.degree. C. in a
solution comprising: 50% formamide, 5x SSC (150 mM NaCl, 15 mM trisodium
citrate), 50 mM sodium phosphate (pH7.6), 5x denhardt's solution, 10%
dextran sulfate, and 20 micrograms/ml of denatured, sheared salmon sperm
DNA, followed by washing the hybridization support in 0.1 x SCC at about
65.degree. C, Hybridization and wash conditions are well known and
Sambrook, et al., Molecular Cloning: A Laboratory Manual , Second Edition,
cold Spring harbor, N.Y., (1989), particularly Chapter 11 therein.
Solution hybridization may also be use with the polynucleotide sequences
provided by the invention.
The invention also provides a polynucleotide consisting of or comprising a
polynucleotide sequences obtained by screening an appropriate library
containing the complete gene for a polynucleotide sequence set forth in
SEQ ID NO:1 under stringent hybridization conditions with a probe having
the said polynucleotide sequence. Fragments useful for obtaining such a
polynucleotide include, for example, probes and primers fully described
elsewhere herein.
As discussed elsewhere herein regarding polynucleotide assays of the
invention, for instance, the polynucleotides of the invention, may be used
as a Hybridization prove for RNA, cDNA and genomic DNA to isolate
full-length cDNAs and genomic clones encoding ups (ugc) and to isolate
cFNA and genomic clones of other genes that have a high identity,
particularly high sequence identity, to the ups (ugc) gene. Such probes
generally will comprise at least 15 nucleotide residues or base pairs and
may have at least 50 nucleotide residues or base pairs Particularly
preferred probes will have at least 20 nucleotide residues or base pairs
and will have less than 30 nucleotide residues or base pairs.
A coding region of a ups (ugc) gene may be isolated by screening using a
DNA sequence provided in table 1 [SEQ ID NO:1] to synthesize an
oligonucleotide probe. A labeled oligonucleotide having a sequence
complementary to that of a gene of the invention is then used to screen a
library of cDNA, genomic DNA or mRNA to determine which members of the
library the probe hybridizes to.
There are several methods available and well known to those skilled in the
art to obtain full-length DNAs, or extend short DNAs, for example those
based on the method of Rapid Amplification of cDNA ends (RACE) (see, for
example, Frohman, et al., PNAS USA 85: 8998-9002, 1988). Recent
modifications of the technique, exemplified by the Marathon.TM. technology
(Clontech Laboratories Inc.) for example, have significantly simplified
the search for longer cDNAs. In the Marathon.TM. technology, cDNAs have
been prepared from mTNA extracted from a chosen tissue and an `adaptor`
sequence ligated onto each end. Nucleic acid amplification (PRC) is then
carried out to amplify the "missing" 5' end of the DNA using a combination
of gene specific and adaptor specific oligonucleotide primers. The PCR
reaction is then repeated using "nested"primers, that is, primers designed
to anneal within the amplified product (typically an adaptor specific
primer that anneals further 3' in the adaptor sequence and a gene specific
primer that anneals further 5' in the selected gene sequence). The
products of this reaction can then be analyzed by DNA sequencing and a
full-length DNA constructed either by joining the product directly to the
existing DNA to give a complete sequence, or carrying out a separate
full-length PCR using the new sequence information for the 5' design.
The polynucleotides and polypeptides of the invention may be employed, for
example, as research reagents and materials for discovery of treatments of
and diagnostics for diseases, particularly human diseases, as further
discussed herein relating to polynucleotide assays.
The polynucleotides of the invention that are oligonucleotide derived from
a sequence of Table 1 [SEQ ID NO:1 or 2] may be used in the processes
herein as described , but preferably for PCR, to determine whether or not
the polynucleotides identified herein in whole or in part are transcribed
in bacteria in infected tissue. It is recognized that such sequences will
also have utility in diagnosis of the stage of infection and type of
infection the pathogen has attained.
The invention also provides polynucleotides that encode a polypeptide that
is the mature protein plus additional amino or carboxyl-terminal amino
acids, or amino acids interior to the mature polypeptide (when the mature
form has more than one polypeptide chain, for instance). Such sequences
may play a role in processing of a protein from precursor to a mature
form, may allow protein transport, may lengthen or shorten protein
half-life or may facilitate manipulation of a protein for assay or
production, among other things. As generally is the case in vivo, the
additional amino acids may be processed away from the mature protein by
cellular enzymes.
For each and every polynucleotide of the invention there is provided a
polynucleotide complementary to it. It is preferred that these
complementary polynucleotides are fully complementary to each
polynucleotide with which they are complementary.
A precursor protein, having a mature form of the polypeptide fused to one
or more prosequences may be an inactive form of the polypeptide. When
prosequences are removed such inactive precursors generally are activated.
Some or all of the prosequences may be removed before activation.
Generally, such precursors are called proproteins.
In addition to the standard A, G, C, T/U representations for nucleotides,
the term "N" may also be used in describing certain polynucleotides of the
invention. "N" means that any of the four DNA or RNA nucleotides may
appear oat such a designated position in the DNA or RNA sequence, except
it is preferred that N is not a nucleic acid that when taken in
combination with adjacent nucleotide positions, when read in the correct
reading frame, would have the effect of generating a premature termination
codon in such reading frame.
In sum, a polynucleotide of the invention may encode a mature protein, a
mature protein plus a leader sequence (which may be referred to as a
preprotein), a precursor of a mature protein having one or more
prosequences that are not the leader sequences of a preprotein, or a
preproprotein, which is a precursor to proprotein, having a leader
sequence and one or more prosequences, which generally are removed during
processing steps that produce active and mature forms of the polypeptide.
Vectors, Host Cells, Expression Systems
The invention also relates to vectors that comprise a polynucleotide or
polynucleotides of the invention, host cells that are genetically
engineered with vectors of the invention and the production of
polypeptides of the invention by recombinant techniques. Cell-free
translation systems can also be employed to produce such proteins using
RNAs derived from the DNA constructs of the invention.
Recombinant polypeptides of the present invention may be prepared by
processes well known in those skilled in the art from genetically
engineered host cells comprising expression systems. Accordingly, in a
further aspect, the present invention relates to expression systems which
comprise a polynucleotide or polynucleotides of the present invention, to
host cells which are genetically engineered with such expression systems,
and to the production of polypeptides of the invention by recombinant
Techniques.
For recombinant production of the polypeptides of the invention, host cells
can be genetically engineered to incorporate expression systems or
portions thereof or polynucleotides of the invention. Introduction of a
polynucleotide into the host cell can be effected by methods described in
many standard laboratory manuals, such as Davis et al., BASIC METHODS IN
MOLECULAR BIOLOGY, (1986)and Sambrook, et al., MOLECULAR CLONING: A
LABORATORY MANUAL, 2nd Ed., Cold spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y. (1989), such as, calcium phosphate transfection,
DEAE-dextran mediated transfection, transfection, microinjection, cationic
lipid-mediated transfection, electroporation, transduction, scrape
loading, ballistic introduction and infection.
Representative examples of appropriate hosts include bacterial cells, such
as cells of streptococci, staphylococci, enterococci E. coli,
streptomyces, eyanbacteria, Bacillus subtilis, and Pseudomonas aeruginosa,
fungal cells, such as cells of yeast, Kluveromyces, Saccharomyces, a
basidiomycete, Candida albicans and Aspergillus; insect cells such as
cells of Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS,
HeLa, C127, 3T3, BHK, 293, CV-1 and Bowes melanoma cells; and plant cells,
such as cells of a gymnosperm or angiosperm.
A great variety of expression system can be used to produce the
polypeptides of the invention. Such vectors include, among others,
chromosomal-, episomal- and virus-derived vectors, for example, vectors
derived from bacterial plasmids, from bacteriophage, from transposons,
from yeast episomes, from insertion elements, from yeast chromosomal
elements, from viruses such as baculoviruses, papova viruses, such as
SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies
viruses, picomaviruses and retroviruses, and vectors derived from
combinations thereof, such as those derived from plasmid and bacteriophage
genetic elements, such as cosmids and phagemids. The expression system
constructs may contain control regions that regulate as well as engender
expression, Generally, any system or vector suitable to maintain,
propagate of express polynucleotides and/or to express a polypeptide in a
host may be used for expression in this regard. The appropriate DNA
sequence may be inserted into the expression system by any of a variety of
well-known and routine techniques, such as, for example, those set forth
in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, (supra).
In recombinant expression systems in eukaryotes, for secretion of a
translated protein into the lumen of the endoplasmic reticulum, into the
periplasmic space or into the extracellular environment, appropriate
secretion signals may be incorporated into the expressed polypeptide.
These signals may be endogenous to the polypeptide or they may be
hererologous signals
Polypeptides of the invention can be recovered and purified from
recombinant cell cultures by well-known methods including ammonium sulfate
or ethanol precipitation, acid extraction, anion or cation exchange
chromatography, phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite chromatography,
and lectin chromatography. Most preferably, high performance liquid
chromatography is employed for purification. Well known techniques for
refolding protein may be employed to regenerate active conformation when
the polypeptide is denatured during isolation and or purification.
Diagnostic, Prognostic, Serotyping and Mutation Assays
This invention is also related to the use of ups (ugc) polynucleotides and
polypeptides of the invention for use a s kidagnostic reagents. Detection
of ups (ugc) polynucleotides and/or polypeptides in a eukaryote,
particularly a mammal, and especially a human, will provide a diagnostic
method for diagnosis of disease, staging of disease or response of an
infectious organism to drugs. Eukaryotes, particularly mammals, and
especially humans, particularly those infected or suspected to be infected
with an organism comprising the ups (ugc) gene or protein, may be detected
at the nucleic acid or amino acid level by a variety of well known
techniques as well a by methods provided herein.
Polypeptides and polynucleotides for prognosis, diagnosis or other analysis
may be obtained from a putatively infected and/or infected individual's
bodily materials. Polynucleotides from any of these sources, particularly
DNA or RNA, may be used directly for detection or may be amplified
enzymatically by using PCR or any other amplification technique prior to
analysis. RNA, particularly mRNA, cDNA and genomic DNA may also be used in
the same ways. Using amplification, characterization of the species and
strain of infectious or resident organism present in an individual, may be
made by an analysis of the genotype of a selected polynucleotide of the
organism. Deletions and insertions can be detected by a change in size of
the amplified product in comparison to a genotype of a reference sequence
selected from a related organism, preferably a different species of the
same genus or a different strain of the same species. Perfectly or
significantly matched sequences can be identified by hybridizing amplified
DNA to labeled ups (ugc) polynucleotide sequences. Perfectly or
significantly matched sequences can be distinguished from imperfectly or
more significantly mismatched duplexes by Dnase or Rnase digestion, for
DNA or RNA respectively, or by detecting differences in melting
temperatures or renaturation kinetics. Polynucleotide sequence differences
may also be detected by alterations in the electrophoretic mobility of
polynucleotide fragments in gels as compared to a reference sequence. This
may be carried out with or without denaturing agents. Polynucleotide
differences may also be detected by direct DNA or RNA sequencing. See, for
example, Nyers et al., Science, 230: 1242(1985). Sequence changes at
specific locations also may be revealed by nuclease protection assays,
such as Rnase, V1 and S1 Protection assay or a chemical cleavage method
See, for example, Cotton et al., Proc. Natl. Acad. Sci., USA, 85:
4397-4401 (1985).
In another embodiment, an array of oligonucleotide probes comprising ups
(ugc) nucleotide sequence or fragments thereof can be constructed to
conduct efficient screening of, for example, genetic mutations,
stereotype, taxonomic classification of identification. Array technology
methods are well known and have general applicability and can be used to
address a variety of questions in molecular genetics including gene
expression, genetic linkage, and genetic variability (see, for example,
Chee et al., Science, 274: 610(1996)).
Thus in another aspect, the present invention relates to a diagnostic kit
which comprises: a polynucleotide of the present invention, preferably the
nucleotide sequence of SEQ ID NO:1, or a fragment thereof; (b) a
nucleotide sequence complementary to that of (a); (c) a polypeptide of the
present invention, preferably the polypeptide of SEQ ID NO:2 or a fragment
thereof; or (d) an antibody to a polypeptide of the present invention,
preferably to the polypeptide of SEQ ID NO:2.
It will be appreciated that in any such kit, (a), (b), (c), or (d) may
comprise a substantial component. Such a kit will be of use in diagnosing
a disease or susceptibility to a Disease, among others.
This invention also relates to the use of polynucleotides of the present
invention as diagnostic reagents. Detection of a mutated from of a
polynucleotide of the invention, preferable, SEQ ID NO:1, which is
associated with a disease or pathogenicity will provide a diagnostic tool
that can add to, or define, a diagnosed of a disease, a prognosis of a
course of disease, a determination of a stage of disease, or a
susceptibility to a disease, which results from underexpression,
overexpression or altered expression of the polynucleotide. Organisms,
particularly infectious organisms, carrying mutations in such
polynucleotide may be detected at the polynucleotide level by a variety of
techniques, such as those described elsewhere herein.
The nucleotide sequences of the present invention are also valuable for
organism chromosome identification. The sequence is specifically targeted
to, and can hybridize with, a particular location on an organism's
chromosome, particularly to a Pseudomonas aeruginosa chromosome. The
mapping of relevant sequences to chromosomes according to the present
invention may be an important step in correlating those sequences with
pathogenic potential and/or an ecological niche of an organism and/or drug
resistance of an organism, as well as the essentially of the gene to the
organism. Once a sequence has been mapped to precise chromosomal location,
the physical position of the sequence on the chromosome can be correlated
with genetic map data. Such data may be found on one in a sequence
database. The relationship between genes and diseases that have been
mapped to the same chromosomal region are then identified through known
genetic methods, for example, through linkage analysis (coinheritance of
physically adjacent genes) or mating studies, such as by conjugation.
The differences in a polynucleotide and/or polypeptide sequence between
organisms possessing a first phenotype and organisms possessing a
different, second different phenotype can also be determined. If a
mutation is observed in some or all organisms possessing the first
phenotype but not n any organisms possessing the second phenotype, then
the mutation is likely to be the causative agent of the first phenotype.
Cells from an organism carrying mutations or polymorphisms (allelic
variations) in a polynucleotide and/or polypeptide of the invention may
also be detected at the polynucleotide or polypeptide level by a variety
of techniques, to allow for serotyping, for example. For example, RT-PCR
can be used to detect mutations in the RNA. It is particularly preferred
to use RT-PCR in conjunction with automated detection systems, such as,
for example, GeneScan. RNA, cDNA or genomic DNA may also be used for the
same purpose, PCR. As an example, PCR primers complementary to a
polynucleotide encoding ups (ugc) polypeptide can be used to identify and
analyze mutations.
These primers may be used for, among other things, amplifying ups (ugc) DNA
and/or RNA isolated from a sample derived from an individual, such as a
bodily material. The primers may be used to amplify a polynucleotide
isolated from an infected individual, such that the polynucleotide may
then be subject to various techniques for elucidation of the
polynucleotide sequence. In this way, mutations in the polynucleotide
sequence may be detected and used to diagnose and/or prognose the
infection or its stage or course, or to serotype and/or classify the
infectious agent.
The invention further provides a process for diagnosing, disease,
preferably bacterial infections, more preferably infections caused by
Pseudomonas aeruginosa, comprising determining from a sample derived from
an individual, such as a bodily material, an increased level of expression
of polynucleotide having a sequence of Table 1 [SEQ ID NO:1]. Increased or
decreased expression of a ups (ugc) polynucleotide can be measured using
any on of the methods well known in the art for the quantitation of
polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNase
protection, Northern blotting, spectrometry and other hybridization
methods.
In addition, a diagnostic assay in accordance with the invention for
detecting over-expression of ups (ugc) polypeptide compared to normal
control tissue samples may be used to detect the presence of an infection,
for example. Assay techniques that can be used to determine levels of a
ups (ugc) polypeptide, in a sample derived from a host, such as a bodily
material, are well-known to those of skill in the art. Such assay methods
include radioimmunoassays, competitive-binding assays, Western Blot
analysis, antibody sandwich assays, antibody detection and ELISA assays.
Differential Expression
The polynucleotides and polynucleotides of the invention may be used as
reagents for differential screening methods. There are many differential
screening and differential display methods known in the art in which the
polynucleotides and polypeptides of the invention may be used. For
example, the differential display technique is described by Chuang et al.,
J. Bacteriol. 175:2026-2036 (1993). This method identifies those genes
which are expressed in an organism by identifying mRNA present using
randomly-primed RT-PCR. By comparing pre-infection and post infection
profiles, genes up and down regulated during infection can be identified
and the RT-PCR product sequenced and matched to ORF "unknowns."
In Vivo Expression Technology (IVET) is described by Camilli et al., Proc.
Nat'l. Acad. Sci. USA. 91:2634-2638 (1994). IVET identifies genes
up-regulated during infection when compared to laboratory cultivation,
implying an important role in infection. ORF's identified by this
technique are implied to have a significant role in infection
establishment and/or maintenance. In this technique random chromosomal
fragments of target organism are cloned upstream of a promoter-less
recombinase gene in a plasmid vector. This construct is introduced into
the target organism which carries an antibiotic resistance gene flanked by
resolvase sites. Growth in the presence of the antibiotic removes from the
population those fragments cloned into the plasmid vector capable of
supporting transcription of the recombinase gene and therefore have caused
loss of antibiotic resistance. The resistant pool is introduced into a
host and at various times after infection bacteria may be recovered and
assessed for the presence of antibiotic resistance. The chromosomal
fragment carried by each antibiotic sensitive bacterium should carry a
promoter or portion of a gene normally upregulated during infection.
Sequencing upstream of the recombinase gene allows identification of the
up regulated gene.
RT-PCR may also be used to analyze gene expression patterns. For RT PCR
using the polynucleotides of the invention, messenger RNA is isolated from
bacterial infected tissue, e.g., 48 hour murine lung infections, and the
amount of each mRNA species assessed by reverse transcription of the RNA
sample primed with random hexanucleotides followed by PCR with gene
specific primer pairs. The determination of the presence and amount of a
particular mRNA species by quantification of the resultant PCR product
provides information on the bacterial genes which are transcribed in the
infected tissue. Analysis of gene transcription can be carried out at
different times of infection to gain a detailed knowledge of gene
regulation in bacterial pathogenesis allowing for a clearer understanding
of which gene products represent targets for screens for antibacterials.
Because of the gene specific nature of the PCR primers employed it should
be understood that the bacterial mRNA preparation need not be free of
mammalian RNA. This allows the investigator to carry out a simple and
quick RNA preparation from infected tissue to obtain bacterial mRNA
species which are very short lived in the bacterium (in the order of 2
minute halflives). Optimally the bacterial mRNA is prepared from infected
murine lung tissue by mechanical disruption in the presence of TRIzole
(GIBCO-BRL) for very short periods of time, subsequent processing
according to the manufacturers of TRIzole reagent and DNAase treatment to
remove contaminating DNA. Preferably the process is optimized by finding
those conditions which give a maximum amount of Pseudomonas aeruginosa 16S
ribosomal RNA as detected by probing Northerns with a suitably labeled
sequence specific oligonucleotide probe. Typically a 5' dye labeled primer
is used in each PCR primer pair in a PCR reaction which is terminated
optimally between 8 and 25 cycles. The PCR products are separated on 6%
polyacrylamide gels with detection and quantification using GeneScanner
(manufactured by ABI).
Gridding and Polynucleotide Subtraction
Methods have been described for obtaining information about gene expression
and identity using so called "high density DNA arrays" or grids. See,
e.g., M. Chee et al., Science, 274:610-614 (1996) and other references
cited therein. Such gridding assays have been employed to identify certain
novel gene sequences, referred to as Expressed Sequence Tags (EST) (Adams
et a., Science, 252:1651-1656 (1991)). A variety of techniques have also
been described for identifying particular gene sequences on the basis of
their gene products. For example, see International Patent Application No.
WO91/07087, published May 30, 1991. In addition, methods have been
described for the amplification of desired sequences. For example, see
International Patent Application No. WO91/17271, published Nov. 14, 1991.
The polynucleotides of the invention may be used as components of
polynucleotide arrays, preferably high density arrays or grids. These high
density arrays are particularly useful for diagnostic and prognostic
purposes. For example, a set of spots each comprising a different gene,
and further comprising a polynucleotide or polynucleotides of the
invention, may be used for probing, such as using hybridization or nucleic
acid amplification, using a probes obtained or derived from a bodily
sample, to determine the presence of a particular polynucleotide sequence
or related sequence in an individual. Such a presence may indicate the
presence of a pathogen, particularly Pseudomonas aeruginosa, and may be
useful in diagnosing and/or prognosing disease or a course of disease. A
grid comprising a number of variants of the polynucleotide sequence of SEQ
ID NO:1 are preferred. Also preferred is a comprising a number of variants
of a polynucleotide sequence encoding the polypeptide sequence of SEQ ID
NO:2.
Antibodies
The polypeptides and polynucleotides of the invention or variants thereof,
or cells expressing the same can be used as immunogens to produce
antibodies immunospecific for such polypeptides or polynucleotides
respectively.
In certain preferred embodiments of the invention there are provided
antibodies against ups (ugc) polypeptides or polynucleotides.
Antibodies generated against the polypeptides or polynucleotides of the
invention can be obtained by administering the polypeptides and/or
polynucleotides of the invention, or epitope-bearing fragments of either
or both, analogues of either or both, or cells expressing either or both,
to an animal, preferably a nonhuman, using routine protocols. For
preparation of monoclonal antibodies, any technique known in the art that
provide antibodies produced by continuous cell line cultures can be used.
Examples include various techniques, such as those in Kohler, G. and
Milstein, C., Nature 256: 495-497 (1975); Kozbor et al., Immunology Today
4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER
THERAPY, Alan R. Liss, Inc. (1985).
Techniques for the production of single chain antibodies (U.S. Pat. No.
4,946,778) can be adapted to produce single chain antibodies to
polypeptides or polynucleotides of this invention. Also, transgenic mice,
or other organisms such as other mammals, may be used to express humanized
antibodies immunospecific to the polypeptides or polynucleotides of the
invention.
Alternatively, phage display technology may be utilized to select antibody
genes with binding activities towards a polypeptide of the invention
either from repertoires of PCR amplified v-genes of lymphocytes from
humans screened for possessing anti-ups (ugc) or from naive libraries
(McCafferty, et al., (1990), Nature 348, 552-554; Marks, et al., (1992)
Biotechnology 10, 779-783). The affinity of these antibodies can also be
improved by, for example, chain shuffling (Clackson et al., (1991) Nature
352: 628).
The above-described antibodies may be employed to isolate or to identify
clones expressing the polypeptides or polynucleotides of the invention to
purify the polypeptides or polynucleotides by, for example, affinity
chromatography.
Thus, among others, antibodies against ups (ugc)-polypeptide or ups
(ugc)-polynucleotide may be employed to treat infections, particularly
bacterial infections.
Polypeptide variants include antigenically, epitopically or immunologically
equivalent variants form a particular aspect of this invention.
A polypeptide or polynucleotide of the invention, such as an antigenically
or immunologically equivalent derivative or a fusion protein of the
polypeptide is used as an antigen to immunize a mouse or other animal such
as a rat or chicken. The fusion protein may provide stability to the
polypeptide. The antigen may be associated, for example by conjugation,
with an immunogenic carrier protein for example bovine serum albumin,
keyhole limpet haemocyanin or tetanus toxoid. Alternatively, a multiple
antigenic polypeptide comprising multiple copies of the polypeptide, or an
antigenically or immunologically equivalent polypeptide thereof may be
sufficiently antigenic to improve immunogenicity so as to obviate the use
of a carrier.
Preferably, the antibody or variant thereof is modified to make it less
immunogenic in the individual. For example, if the individual is human the
antibody may most preferably be "humanized," where the complimentarity
determining region or regions of the hybridoma-derived antibody has been
transplanted into a human monoclonal antibody, for example as described in
Jones et al. (1986), Nature 321, 522-525 or Tempest et al., (1991)
Biotechnology 9, 266-273.
In accordance with an aspect of the invention, there is provided the use of
a polynucleotide of the invention for therapeutic or prophylactic
purposes, in particular genetic immunization. Among the particularly
preferred embodiments of the invention are naturally occurring allelic
variants of ups (ugc) polynucleotides and polypeptides encoded thereby.
The use of a polynucleotide of the invention in genetic immunization will
preferably employ a suitable delivery method such as direct injection of
plasmid DNA into muscles (Wolff et al., Hum Mol Genet (1992) 1: 363,
Manthorpe et al., Hum. Gene Ther. (1983) 4: 419), delivery of DNA
complexed with specific protein carriers (Wu et al., J Biol Chem. (1989)
264: 16985), coprecipitation of DNA with calcium phosphate (Benvenisty &
Reshef, PNAS USA, (1986) 83: 9551), encapsulation of DNA in various forms
of liposomes (Kaneda et al., Science (1989) 243: 375), particle
bombardment (Tang et al., Nature (1992) 356:152, Eisenbraun et al., DNA
Cell Biol (1993) 12: 791) and in vivo infection using cloned retroviral
vectors (Seeger et al., PNAS USA (1984) 81: 5849).
Antagonists and Agonists--Assays and Molecules
Polypeptides and polynucleotides of the invention may also be used to asses
the binding of small molecule substrates and ligands in, for example,
cells, cell-free preparations, chemical libraries, and natural product
mixtures. These substrates and ligands may be natural substrates and
ligands or may be structural or functional mimetics. See, e.g., Coligan et
al., Current Protocols in Immunology 1(2): Chapter 5 (1991).
Polypeptides and polynucleotides of the present invention are responsible
for many biological functions, including many disease states, in
particular the Diseases hereinbefore mentioned. It is therefore desirable
to devise screening methods to identify compounds which stimulate or which
inhibit the function of the polypeptide or polynucleotide. Accordingly, in
a further aspect, the present invention provides for a method of screening
compounds to identify those which stimulate or which inhibit the function
of a polypeptide or polynucleotide of the invention, as well as related
polypeptides and polynucleotides. In general, agonists or antagonists may
be employed for therapeutic and prophylactic purposes for such Diseases as
hereinbefore mentioned. Compounds may be identified from a variety of
sources, for example, cells, cell-free preparations, chemical libraries,
and natural product mixtures. Such agonists, antagonists or inhibitors
so-identified may be natural or modified substrates, ligands, receptors,
enzymes, etc., as the case may be, of ups (ugc) polypeptides and
polynucleotides; or may be structural or functional mimetics thereof (see
Coligan et al., Current Protocols in Immunology 1(2):Chapter 5 (1991)).
The screening methods may simply measure the binding of a candidate
compound to the polypeptide or polynucleotide, or to cells or membranes
bearing the polypeptide or polynucleotide, or a fusion protein of the
polypeptide by means of a label directly or indirectly associated with the
candidate compound. Alternatively, the screening method may involve
competition with a labeled competitor. Further, these screening methods
may test whether the candidate compound results in a signal generated by
activation or inhibition of the polypeptide or polynucleotide, using
detection systems appropriate to the cells comprising the polypeptide or
polynucleotide. Inhibitors of activation are generally assayed in the
presence of a known agonist and the effect on activation by the agonist by
the presence of the candidate compound is observed. Constitutively active
polypeptide and/or constitutively expressed polypeptides and
polynucleotides may be employed in screening methods for inverse agonists
or inhibitors, in the absence of an agonist or inhibitor, by testing
whether the candidate compound results in inhibition of activation of the
polypeptide or polynucleotide, as the case may be. Further, the screening
methods may simply comprise the steps of mixing a candidate compound with
a solution containing a polypeptide or polynucleotide of the present
invention, to form a mixture, measuring ups (ugc) polypeptide and/or
polynucleotide activity in the mixture, and comparing the ups (ugc)
polypeptide and/or polynucleotide activity of the mixture to a standard.
Fusion proteins, such as those made from Fc portion and ups (ugc)
polypeptide, as hereinbefore described, can also be used for
high-throughput screening assays to identify antagonists of the
polypeptide of the present invention, as well as of phylogenetically and
and/or functionally related polypeptides (see D. Bennett et al., J Mol
Recognition, 8:52-58 (1995); and K. Johanson et al., J Biol Chem,
270(16):9459-9471 (1995)).
The polynucleotides, polypeptides and antibodies that bind to and/or
interact with a polypeptide of the present invention may also be used to
configure screening methods for detecting the effect of added compounds on
the production of mRNA and/or polypeptide in cells. For example, an ELISA
assay may be constructed for measuring secreted or cell associated levels
of polypeptide using monoclonal and polyclonal antibodies by standard
methods known in the art. This can be used to discover agents which may
inhibit or enhance the production of polypeptide (also called antagonist
or agonist, respectively) from suitably manipulated cells or tissues.
The invention also provides a method of screening compounds to identify
those which enhance (agonist) or block (antagonist) the action of ups
(ugc) polypeptides or polynucleotides, particularly those compounds that
are bacteristatic and/or bactericidal. The method of screening may involve
high-throughput techniques. For example, to screen for agonists or
antagonists, a synthetic reaction mix, a cellular compartment, such as a
membrane, cell envelope or cell wall, or a preparation of any thereof,
comprising ups (ugc) polypeptide and a labeled substrate or ligand or such
polypeptide is incubated in the absence or the presence of a candidate
molecule that may be a ups (ugc) agonist or antagonist. The ability of the
candidate molecule to agonize or antagonize the ups (ugc) polypeptide is
reflected in decreased binding of the labeled ligand or decreased
production of product from such substrate. Molecules that bind
gratuitously, i.e., without inducing the effects of ups (ugc) polypeptide
are most likely to be good antagonists. Molecules that bind well and, as
the case may be, increase the rate of product production from substrate,
increase signal transduction, or increase chemical channel activity are
agonists. Detection of the rate or level of, as the case may be,
production of product from substrate, signal transduction, or chemical
channel activity may be enhanced by using a reporter system. Reporter
systems that may be useful in this regard include but are not limited to
colorimetric, labeled substrate converted into product, a reporter gene
that is responsive to changes in ups (ugc) polynucleotide or polypeptide
activity, and binding assays known in the art.
Polypeptides of the invention may be used to identify membrane bound or
soluble receptors, if any, for such polypeptide, through standard receptor
binding techniques known in the art. These techniques include, but are not
limited to, ligand binding and crosslinking assays in which the
polypeptide is labeled with a radioactive isotope (for instance, .sup.125
I), chemically modified (for instance, biotinylated), or fused to a
peptide sequence suitable for detection or purification, and incubated
with a source of the putative receptor (e.g., cells, cell membranes, cell
supernatants, tissue extracts, bodily materials). Other methods include
biophysical techniques such as surface plasmon resonance and spectroscopy.
These screening methods may also be used to identify agonists and
antagonists of the polypeptide which compete with the binding of the
polypeptide to its receptor(s), if any. Standard methods for conducting
such assays are well understood in the art.
The fluorescence polarization value for a fluorescently-tagged molecule
depends on the rotational correlation time or tumbling rate. Protein
complexes, such as formed by ups (ugc) polypeptide associating with
another ups (ugc) polypeptide or other polypeptide, labeled to comprise a
fluorescently-labeled molecule will have higher polarization values than a
fluorescently labeled monomeric protein. It is preferred that this method
be used to characterize small molecules that disrupt polypeptide
complexes.
Fluorescence energy transfer may also be used characterize small molecules
that interfere with the formation of ups (ugc) polypeptide dimers,
trimers, tetramers or higher order structures, or structures formed by ups
(ugc) polypeptide bound to another polypeptide. Ups (ugc) polypeptide can
be labeled with both a donor and acceptor fluorophore. Upon mixing of the
two labeled species and excitation of the donor fluorophore, fluorescence
energy transfer can be detected by observing fluorescence of the acceptor.
Compounds that block dimerization will inhibit fluorescence energy
transfer.
Surface plasmon resonance can be used to monitor the effect of small
molecules on ups (ugc) polypeptide self-association as well as an
association of ups (ugc) polypeptide and another polypeptide or small
molecule. ups (ugc) polypeptide can be coupled to a sensor chip at low
site density such that covalently bound molecules will be monomeric.
Solution protein can then passed over the ups (ugc) polypeptide-coated
surface and specific binding can be detected in real-time by monitoring
the change in resonance angle caused by a change in local refractive
index. This technique can be used to characterize the effect of small
molecules on kinetic rates and equilibrium binding constants for ups (ugc)
polypeptide self-association as well as an association of ups (ugc)
polypeptide and another polypeptide or small molecule.
A scintillation proximity assay may be used to characterize the interaction
between an association of ups (ugc) polypeptide with another ups (ugc)
polypeptide or a different polypeptide ups (ugc) polypeptide can be
coupled to a scintillation-filled bead. Addition of radio-labeled ups
(ugc) polypeptide results in binding where the radioactive source molecule
is in close proximity to the scintillation fluid. Thus, signal is emitted
upon ups (ugc) polypeptide binding and compounds that prevent ups (ugc)
polypeptide self-association or an association of ups (ugc) polypeptide
and another polypeptide or small molecule will diminish signal.
ICS biosensors have been described by AMBRI (Australian Membrane
Biotechnology Research Institute). They couple the self-association of
macromolecules to the closing of gramacidin-facilitated ion channels in
suspended membrane bilayers and hence to a measurable change in the
admittance (similar to impedence) of the biosensor. This approach is
linear over six decades of admittance change and is ideally suited for
large scale, high through-put screening of small molecule combinatorial
libraries.
In other embodiments of the invention there are provided methods for
identifying compounds which bind to or otherwise interact with and inhibit
or activate an activity or expression of a polypeptide and/or
polynucleotide of the invention comprising: contacting a polypeptide
and/or polynucleotide of the invention with a compound to be screened
under conditions to permit binding to or other interaction between the
compound and the polypeptide and/or polynucleotide to assess the binding
to or other interaction with the compound, such binding or interaction
preferably being associated with a second component capable of providing a
detectable signal in response to the binding or interaction of the
polypeptide and/or polynucleotide with the compound; and determining
whether the compound binds to or otherwise interacts with and activates or
inhibits an activity or expression of the polypeptide and/or
polynucleotide by detecting the presence or absence of a signal generated
from the binding or interaction of the compound with the polypeptide
and/or polynucleotide.
Another example of an assay for ups (ugc) agonists is a competitive assay
that combines ups (ugc) and a potential agonist with ups (ugc)-binding
molecules, recombinant ups (ugc) binding molecules, natural substrates or
ligands, or substrate or ligand mimetics, under appropriate conditions for
a competitive inhibition assay. ups (ugc) can be labeled, such as by
radioactivity or a colorimetric compound, such that the number of ups
(ugc) molecules bound to a binding molecule or converted to product can be
determined accurately to assess the effectiveness of the potential
antagonist.
Potential antagonists include, among others, small organic molecules,
peptides, polypeptides and antibodies that bind to a polynucleotide and/or
polypeptide of the invention and thereby inhibit or extinguish its
activity or expression. Potential antagonists also may be small organic
molecules, a peptide, a polypeptide such as a closely related protein or
antibody that binds the same sites on a binding molecule, such as a
binding molecule, without inducing ups (ugc)-induced activities, thereby
preventing the action or expression or ups (ugc) polypeptides and/or
polynucleotides by excluding ups (ugc) polypeptides and/or polynucleotides
from binding.
Potential antagonists include a small molecule that binds to and occupies
the binding site of the polypeptide thereby preventing binding to cellular
binding molecules, such that normal biological activity is prevented.
Examples of small molecules include but are not limited to small organic
molecules, peptides or peptide-like molecules. Other potential antagonists
include antisense molecules (see Okano, J. Neurochem. 56: 560 (1991);
OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC
Press, Boca Raton, Fla. (1988), for a description of these molecules).
Preferred potential antagonists include compounds related to and variants
of ups (ugc).
Other examples of potential polypeptide antagonists include antibodies or,
in some cases, oligonucleotides or proteins which are closely related to
the ligands, substrates, receptors, enzymes, etc., as the case may be, of
the polypeptide, e.g., a fragment of the ligands, substrates, receptors,
enzymes, etc.; or small molecules which bind to the polypeptide of the
present invention but do not elicit a response, so that the activity of
the polypeptide is prevented.
Certain of the polypeptide of the invention are biomimetics, functional
mimetics of the natural ups (ugc) polypeptide. These functional mimetics
may be used for, among other things, antagonizing the activity of ups
(ugc) polypeptide or as a antigen or immunogen in a manner described
elsewhere herein. Functional mimetics of the polypeptides of the invention
include but are not limited to truncated polypeptides. For example,
preferred functional mimetics include, a polypeptide comprising the
polypeptide sequence set forth in SEQ ID NO:2 lacking 20, 30, 40, 50, 60,
70 or 80 amino- or carboxy-terminal amino acid residues, including fusion
proteins comprising one or more of these truncated sequences.
Polynucleotides encoding each of these functional mimetics may be used as
expression cassettes to express each mimetic polypeptide. It is preferred
that these cassettes comprise 5' and 3' restriction sites to allow for a
convenient means to ligate the cassettes together when desired. It is
further preferred that these cassettes comprise gene expression signals
known in the art or described elsewhere herein.
Thus, in another aspect, the present invention relates to a screening kit
for identifying agonists, antagonists, ligands, receptors, substrates,
enzymes, etc. for a polypeptide and/or polynucleotide of the present
invention; or compounds which decrease or enhance the production of such
polypeptides and/or polynucleotides, which comprises: (a) a polypeptide
and/or a polynucleotide of the present invention; (b) a recombinant cell
expressing a polypeptide and/or polynucleotide of the present invention;
(c) a cell membrane expressing a polypeptide and/or polynucleotide of the
present invention; or (d) antibody to a polypeptide and/or polynucleotide
of the present invention;
which polypeptide is preferably that of SEQ ID NO:2, and which
polynucleotide is preferably that of SEQ ID NO:1.
It will be appreciated that in any such kit, (a), (b), (c) or (d) may
comprise a substantial component.
It will be readily appreciated by the skilled artisan that a polypeptide
and/or polynucleotide of the present invention may also be used in a
method for the structure-based design of an agonist, antagonist or
inhibitor of the polypeptide and/or polynucleotide, by: (a) determining in
the first instance the three-dimensional structure of the polypeptide
and/or polynucleotide, or complexes thereof; (b) deducing the
three-dimensional structure for the likely reactive site(s), binding
site(s) or motif(s) of an agonist, antagonist or inhibitor; (c)
synthesizing candidate compounds that are predicted to bind to or react
with the deduced binding site(s), reactive site(s), and/or motif(s); and
(d) testing whether the candidate compounds are indeed agonists,
antagonists or inhibitors.
It will be further appreciated that this will normally be an iterative
process, and this iterative process may be performed using automated and
computer-controlled steps.
In a further aspect, the present invention provides methods of treating
abnormal conditions such as, for instance, a Disease, related to either an
excess of, an under-expression of, an elevated activity of, or a decreased
activity of ups (ugc) polypeptide and/or polynucleotide.
If the expression and/or activity of the polypeptide and/or polynucleotide
is in excess, several approaches are available. One approach comprises
administering to an individual in need thereof an inhibitor compound
(antagonist) as herein described, optionally in combination with a
pharmaceutically acceptable carrier, in an amount effective to inhibit the
function and/or expression of the polypeptide and/or polynucleotide, such
as, for example, by blocking the binding of ligands, substrates,
receptors, enzymes, etc., or by inhibiting a second signal, and thereby
alleviating the abnormal condition. In another approach, soluble forms of
the polypeptides still capable of binding the ligand, substrate, enzymes,
receptors, etc. in competition with endogenous polypeptide and/or
polynucleotide may be administered. Typical examples of such competitors
include fragments of the ups (ugc) polypeptide and/or polynucleotide.
In a further aspect, the present invention relates to genetically
engineered soluble fusion proteins comprising a polypeptide of the present
invention, or a fragment thereof, and various portions of the constant
regions of heavy or light chains of immunoglobulins of various subclasses
(IgG, IgM, IgA, IgE). Preferred as an immunoglobulin is the constant part
of the heavy chain of human IgG, particularly IgG1, where fusion takes
place at the hinge region. In a particular embodiment, the Fc part can be
removed simply by incorporation of a cleavage sequence which can be
cleaved with blood clotting factor Xa. Furthermore, this invention relates
to processes for the preparation of these fusion proteins by genetic
engineering, and to the use thereof for drug screening, diagnosis and
therapy. A further aspect of the invention also relates to polynucleotides
encoding such fusion proteins. Examples of fusion protein technology can
be found in International Patent Application Nos. WO94/29458 and
WO94/22914.
In still another approach, expression of the gene encoding endogenous ups
(ugc) polypeptide can be inhibited using expression blocking techniques.
This blocking may be targeted against any step in gene expression, but is
preferably targeted against transcription and/or translation. An examples
of a known technique of this sort involve the use of antisense sequences,
either internally generated or separately administered (see, for example,
O'Connor, J Neurochem (1991) 56:560 in Oligodeoxynucleotides as Antisense
Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)).
Alternatively, oligonucleotides which form triple helices with the gene
can be supplied (see, for example, Lee et al., Nucleic Acids Res (1979)
6:3073; Cooney et al., Science (1988) 241:456; Dervan et al., Science
(1991) 251:1360). These oligomers can be administered per se or the
relevant oligomers can be expressed in vivo.
Each of the polynucleotide sequences provided herein may be used in the
discovery and development of antibacterial compounds. The encoded protein,
upon expression, can be used as a target for the screening of
antibacterial drugs. Additionally, the polynucleotide sequences encoding
the amino terminal regions of the encoded protein or Shine-Delgarno or
other translation facilitating sequences of the respective mRNA can be
used to construct antisense sequences to control the expression of the
coding sequence of interest.
The invention also provides the use of the polypeptide, polynucleotide,
agonist or antagonist of the invention to interfere with the initial
physical interaction between a pathogen or pathogens and a eukaryotic,
preferably mammalian, host responsible for sequelae of infection. In
particular, the molecules of the invention may be used: in the prevention
of adhesion of bacteria, in particular gram positive and/or gram negative
bacteria, to eukaryotic, preferably mammalian, extracellular matrix
proteins on in-dwelling devices or to extracellular matrix proteins in
wounds; to block bacterial adhesion between eukaryotic, preferably
mammalian, extracellular matrix proteins and bacterial ups (ugc) proteins
that mediate tissue damage and/or; to block the normal progression of
pathogenesis in infections initiated other than by the implantation of
in-dwelling devices or by other surgical techniques.
In accordance with yet another aspect of the invention, there are provided
ups (ugc) agonists and antagonists, preferably bacteristatic or
bactericidal agonists and antagonists.
The antagonists and agonists of the invention may be employed, for
instance, to prevent, inhibit and/or treat diseases.
Helicobacter pylori (herein "H. pylori") bacteria infect the stomachs of
over one-third of the world's population causing stomach cancer, ulcers,
and gastritis (International Agency for Research on Cancer (1994)
Schistosomes, Liver Flukes and Helicobacter Pylori (International Agency
for Research on Cancer, Lyon, France, http://www.uice.ch/ecp/ecp2904.htm).
Moreover, the International Agency for Research on Cancer recently
recognized a cause-and-effect relationship between H. pylori and gastric
adenocarcinoma, classifying the bacterium as a Group I (definite)
carcinogen. Preferred antimicrobial compounds of the invention (agonists
and antagonists of ups (ugc) polypeptides and/or polynucleotides) found
using screens provided by the invention, or known in the art, particularly
narrow-spectrum antibiotics, should be useful in the treatment of H.
pylori infection. Such treatment should decrease the advent of H.
pylori-induced cancers, such as gastrointestinal carcinoma. Such treatment
should also prevent, inhibit and/or cure gastric ulcers and gastritis.
Vaccines
There are provided by the invention, products, compositions and methods for
assessing ups (ugc) expression, treating disease, assaying genetic
variation, and administering a ups (ugc) polypeptide and/or polynucleotide
to an organism to raise an immunological response against a bacteria,
especially a Pseudomonas aeruginosa bacteria.
Another aspect of the invention relates to a method for inducing an
immunological response in an individual, particularly a mammal which
comprises inoculating the individual with ups (ugc) polynucleotide and/or
polypeptide, or a fragment or variant thereof, adequate to produce
antibody and/or T cell immune response to protect said individual from
infection, particularly bacterial infection and most particularly
Pseudomonas aeruginosa infection. Also provided are methods whereby such
immunological response slows bacterial replication. Yet another aspect of
the invention relates to a method of inducing immunological response in an
individual which comprises delivering to such individual a nucleic acid
vector, sequence or ribozyme to direct expression of ups (ugc)
polynucleotide and/or polypeptide, or a fragment or a variant thereof, for
expressing ups (ugc) polynucleotide and/or polypeptide, or a fragment or a
variant thereof in vivo in order to induce an immunological response, such
as, to produce antibody and/or T cell immune response, including, for
example, cytokine-producing T cells or cytotoxic T cells, to protect said
individual, preferably a human, from disease, whether that disease is
already established within the individual or not. One example of
administering the gene is by accelerating it into the desired cells as a
coating on particles or otherwise. Such nucleic acid vector may comprise
DNA, RNA, a ribozyme, a modified nucleic acid, a DNA/RNA hybrid, a
DNA-protein complex or an RNA-protein complex.
A further aspect of the invention relates to an immunological composition
that when introduced into an individual, preferably a human, capable of
having induced within it an immunological response, induces an
immunological response in such individual to a ups (ugc) polynucleotide
and/or polypeptide encoded therefrom, wherein the composition comprises a
recombinant ups (ugc) polynucleotide and/or polypeptide encoded therefrom
and/or comprises DNA and/or RNA which encodes and expresses an antigen of
said ups (ugc) polynucleotide, polypeptide encoded therefrom, or other
polypeptide of the invention. The immunological response may be used
therapeutically or prophylactically and may take the form of antibody
immunity and/or cellular immunity, such as cellular immunity arising from
CTL or CD4+ T cells.
A ups (ugc) polypeptide or a fragment thereof may be fused and co-protein
or chemical moiety which may or may not by itself produce antibodies, but
which is capable of stabilizing the first protein and producing a fused or
modified protein which will have antigenic and/or immunogenic properties,
and preferably protective properties. Thus fused recombinant protein,
preferably further comprises an antigenic co-protein, such as lipoprotein
D from Hemophilus influenzae, Glutathione-S-transferase (GST) or
beta-galactosidase, or any other relatively large co-protein which
solubilizes the protein and facilitates production and purification
thereof. Moreover, the co-protein may act as an adjuvant in the sense of
providing a generalized stimulation of the immune system of the organism
receiving the protein. The co-protein may be attached to either the amino-
or carboxy-terminus of the first protein.
Provided by this invention are compositions, particularly vaccine
compositions, and methods comprising the polypeptides and/or
polynucleotides of the invention and immunostimulatory DNA sequences, such
as those described in Sato, Y. et al. Science 273: 352 (1996).
Also, provided by this invention are methods using the described
polynucleotide or particular fragments thereof, which have been shown to
encode non-variable regions of bacterial cell surface proteins, in
polynucleotide constructs used in such genetic immunization experiments in
animal models of infection with Pseudomonas aeruginosa. Such experiments
will be particularly useful for identifying protein epitopes able to
provoke a prophylactic or therapeutic immune response. It is believed that
this approach will allow for the subsequent preparation of monoclonal
antibodies of particular value, derived from the requisite organ of the
animal successfully resisting or clearing infection, for the development
of prophylactic agents or therapeutic treatments of bacterial infection,
particularly Pseudomonas aeruginosa infection, in mammals, particularly
humans.
A polypeptide of the invention may be used as an antigen for vaccination of
a host to produce specific antibodies which protect against invasion of
bacteria, for example by blocking adherence of bacteria to damaged tissue.
Examples of tissue damage include wounds in skin or connective tissue
caused, for example, by mechanical, chemical, thermal or radiation damage
or by implantation of indwelling devices, or wounds in the mucous
membranes, such as the mouth, throat, mammary glands, urethra or vagina.
The invention also includes a vaccine formulation which comprises an
immunogenic recombinant polypeptide and/or polynucleotide of the invention
together with a suitable carrier, such as a pharmaceutically acceptable
carrier. Since the polypeptides and polynucleotides may be broken down in
the stomach, each is preferably administered parenterally, including, for
example, administration that is subcutaneous, intramuscular, intravenous,
or intradermal. Formulations suitable for parenteral include aqueous and
non-aqueous sterile injection solutions which may contain anti-oxidants,
buffers, bacteristatic compounds and solutes which render the formulation
isotonic with the bodily fluid, preferably the blood, of the individual;
and aqueous and non-aqueous sterile suspensions which may include
suspending agents or thickening agents. The formulations may be presented
in unit-dose or multi-dose containers, for example, sealed ampoules and
vials and may be stored in a freeze-dried condition requiring only the
addition of the sterile liquid carrier immediately prior to use. The
vaccine formulation may also include adjuvant systems for enhancing the
immunogenicity of the formulation, such as oil-in water systems and other
systems known in the art. The dosage will depend on the specific activity
of the vaccine and can be readily determined by routine experimentation.
While the invention has been described with reference to certain ups (ugc)
polypeptides and polynucleotides, it is to be understood that this covers
fragments of the naturally occurring polypeptides and polynucleotides, and
similar polypeptides and polynucleotides with additions, deletions or
substitutions which do not substantially affect the immunogenic properties
of the recombinant polypeptides or polynucleotides.
Compositions, kits and administration
In a further aspect of the invention there are provided compositions
comprising a ups (ugc) polynucleotide and/or a ups (ugc) polypeptide for
administration to a cell or to a multicellular organism.
The invention also relates to compositions comprising a polynucleotide and
or a polypeptides discussed herein or their agonists or antagonists. The
polypeptides and polynucleotides of the invention may be employed in
combination with a non-sterile carrier or carriers for use with cells,
tissues or organisms, such as a pharmaceutical carrier suitable for
administration to an individual. Such compositions comprise, for instance,
a media additive or a therapeutically effective amount of a polypeptide
and/or polynucleotide of the invention and a pharmaceutically acceptable
carrier or excipient. Such carriers may include, but are not limited to,
saline, buffered saline, dextrose, water, glycerol, ethanol and
combinations thereof. The formulation should suit the mode of
administration. The invention further relates to diagnostic and
pharmaceutical packs and kits comprising one or more containers filled
with one or more of the ingredients of the aforementioned compositions of
the invention.
Polypeptides, polynucleotides and other compounds of the invention may be
employed alone or in conjunction with other compounds, such as therapeutic
compounds.
The pharmaceutical compositions may be administered in any effective,
convenient manner including, for instance, administration by topical,
oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,
subcutaneous, intranasal or intradermal routes among others.
In therapy or as a prophylactic, the active agent may be administered to an
individual as an injectable composition, for example as a sterile aqueous
dispersion, preferably isotonic.
Alternatively the composition may be formulated for topical application for
example in the form of ointments, creams, lotions, eye ointments, eye
drops, ear drops, mouthwash, impregnated dressings and sutures and
aerosols, and may contain appropriate conventional additives, including,
for example, preservatives, solvents to assist drug penetration, and
emollients in ointments and creams. Such topical formulations may also
contain compatible conventional carriers, for example cream or ointment
bases, and ethanol or oleyl alcohol for lotions. Such carriers may
constitute from about 1% to about 98% by weight of the formulation; more
usually they will constitute up to about 80% by weight of the formulation.
In a further aspect, the present invention provides for pharmaceutical
compositions comprising a therapeutically effective amount of a
polypeptide and/or polynucleotide, such as the soluble form of a
polypeptide and/or polynucleotide of the present invention, agonist or
antagonist peptide or small molecule compound, in combination with a
pharmaceutically acceptable carrier or excipient. Such carriers include,
but are not limited to, saline, buffered saline, dextrose, water,
glycerol, ethanol, and combinations thereof. The invention further relates
to pharmaceutical packs and kits comprising one or more containers filled
with one or more of the ingredients of the aforementioned compositions of
the invention. Polypeptides, polynucleotides and other compounds of the
present invention may be employed alone or in conjunction with other
compounds, such as therapeutic compounds.
The composition will be adapted to the route of administration, for
instance by a systemic or an oral route. Preferred forms of systemic
administration include injection, typically by intravenous injection.
Other injection routes, such as subcutaneous, intramuscular, or
intraperitoneal, can be used. Alternative means for systemic
administration include transmucosal and transdermal administration using
penetrants such as bile salts or fusidic acids or other detergents. In
addition, if a polypeptide or other compounds of the present invention can
be formulated in an enteric or an encapsulated formulation, oral
administration may also be possible. Administration of these compounds may
also be topical and/or localized, in the form of salves, pastes, gels, and
the like.
For administration to mammals, and particularly humans, it is expected that
the daily dosage level of the active agent will be from 0.01 mg/kg to 10
mg/kg, typically around 1 mg/kg. The physician in any event will determine
the actual dosage which will be most suitable for an individual and will
vary with the age, weight and response of the particular individual. The
above dosages are exemplary of the average case. There can, of course, be
individual instances where higher or lower dosage ranges are merited, and
such are within the scope of this invention.
In-dwelling devices include surgical implants, prosthetic devices and
catheters, i.e., devices that are introduced to the body of an individual
and remain in position for an extended time. Such devices include, for
example, artificial joints, heart valves, pacemakers, vascular grafts,
vascular catheters, cerebrospinal fluid shunts, urinary catheters,
continuous ambulatory peritoneal dialysis (CAPD) catheters.
The composition of the invention may be administered by injection to
achieve a systemic effect against relevant bacteria shortly before
insertion of an in-dwelling device. Treatment may be continued after
surgery during the in-body time of the device. In addition, the
composition could also be used to broaden perioperative cover for any
surgical technique to prevent bacterial wound infections, especially
Pseudomonas aeruginosa wound infections.
Many orthopedic surgeons consider that humans with prosthetic joints should
be considered for antibiotic prophylaxis before dental treatment that
could produce a bacteremia. Late deep infection is a serious complication
sometimes leading to loss of the prosthetic joint and is accompanied by
significant morbidity and mortality. It may therefore be possible to
extend the use of the active agent as a replacement for prophylactic
antibiotics in this situation.
In addition to the therapy described above, the compositions of this
invention may be used generally as a wound treatment agent to prevent
adhesion of bacteria to matrix proteins exposed in wound tissue and for
prophylactic use in dental treatment as an alternative to, or in
conjunction with, antibiotic prophylaxis.
Alternatively, the composition of the invention may be used to bathe an
indwelling device immediately before insertion. The active agent will
preferably be present at a concentration of 1 .mu.g/ml to 10 mg/ml for
bathing of wounds or indwelling devices.
A vaccine composition is conveniently in injectable form. Conventional
adjuvants may be employed to enhance the immune response. A suitable unit
dose for vaccination is 0.5-5 microgram/kg of antigen, and such does is
preferably administered 1-3 times and with an interval of 1-3 weeks. With
the indicated dose range, no adverse toxicological effects will be
observed with the compounds of the invention which would preclude their
administration to suitable individuals.
Sequence Databases, Sequences in a Tangible Medium, and Algorithms
Polynucleotide and polypeptide sequences form a valuable information
resource with which to determine their 2- and 3-dimensional structures as
well as to identify further sequences of similar homology. These
approaches are most easily facilitated by storing the sequence in a
computer readable medium and then using the stored data in a known
macromolecular structure program or to search a sequence database using
well known searching tools, such as GCC.
The polynucleotide and polypeptide sequences of the invention are
particularly useful as components in databases useful for search analyses
as well as in sequence analysis algorithms. As used in this section
entitled "Sequence Databases, Sequences in a Tangible Medium, and
Algorithms," and in claims related to this section, the terms
"polynucleotide of the invention" and "polynucleotide sequence of the
invention" mean any detectable chemical or physical characteristic of a
polynucleotide of the invention that is or may be reduced to or stored in
a tangible medium, preferably a computer readable form. For example,
chromatographic scan data or peak data, photographic data or scan data
therefrom, called bases, and mass spectrographic data. As used in this
section entitled Databases and Algorithms and in claims related thereto,
the terms "polypeptide of the invention" and "polypeptide sequence of the
invention" mean any detectable chemical or physical characteristic of a
polypeptide of the invention that is or may be reduced to or stored in a
tangible medium, preferably a computer readable form. For example,
chromatographic scan data or peak data, photographic data or scan data
therefrom, and mass spectrographic data.
The invention provides a computer readable medium having stored thereon
polypeptide sequences of the invention and/or polynucleotide sequences of
the invention. For example, a computer readable medium is provided
comprising and having stored thereon a member selected from the group
consisting of: a polynucleotide comprising the sequence of a
polynucleotide of the invention; a polypeptide comprising the sequence of
a polypeptide sequence of the invention; a set of polynucleotide sequences
wherein at least one of the sequences comprises the sequence of a
polynucleotide sequence of the invention; a set of polypeptide sequences
wherein at least one of the sequences comprises the sequence of a
polypeptide sequence of the invention; a data set representing a
polynucleotide sequence comprising the sequence of polynucleotide sequence
of the invention; a data set representing a polynucleotide sequence
encoding a polypeptide sequence comprising the sequence of a polypeptide
sequence of the invention; a polynucleotide comprising the sequence of a
polynucleotide sequence of the invention; a polypeptide comprising the
sequence of a polypeptide sequence of the invention; a set of
polynucleotide sequences wherein at least one of the sequences comprises
the sequence of a polynucleotide sequence of the invention; a set of
polypeptide sequences wherein at least one of said sequences comprises the
sequence of a polypeptide sequence of invention; a data set representing a
polynucleotide sequence comprising the sequence of a polynucleotide
sequence of the invention; a data set representing a polynucleotide
sequence encoding a polypeptide sequence comprising the sequence of a
polypeptide sequence of the invention. The computer readable medium can be
any composition of matter used to store information of data, including,
for example, commercially available floppy disks, tapes, chips, hard
drives, compact disks, and video disks.
Also provided by the invention are methods for the analysis of character
sequences or strings, particularly genetic sequences or encoded genetic
sequences. Preferred methods of sequence analysis include, for example,
methods of sequence homology analysis, such as identity and similarity
analysis, RNA structure analysis, sequence assembly, cladistic analysis,
sequence motif analysis, open reading frame determination, nucleic acid
base calling, nucleic acid base trimming, and sequence chromatogram peak
analysis.
A computer based method is provided for performing homology identification.
This method comprises the steps of providing a first polynucleotide
sequence comprising the sequence a polynucleotide of the invention in a
computer readable medium; and comparing said first polynucleotide sequence
to at least one second polynucleotide or polypeptide sequence to identify
homology.
A computer based method is also provided for performing homology
identification, said method comprising the steps of: providing a first
polypeptide sequence comprising the sequence of a polypeptide of the
invention in a computer readable medium; and comparing said first
polypeptide sequence to at least one second polynucleotide or polypeptide
sequence to identify homology.
A computer based method is still further provided for polynucleotide
assembly, said method comprising the steps of: providing a first
polynucleotide sequence comprising the sequence of a polynucleotide of the
invention in a computer readable medium; and screening for at least one
overlapping region between said first polynucleotide sequence and at least
one second polynucleotide or polypeptide sequence.
A computer based method is still further provided for polynucleotide
assembly, said method comprising the steps of: providing a first
polypeptide sequence comprising a polypeptide of the invention in a
computer readable medium; and screening for at least one overlapping
region between said first polypeptide sequence and at least one second
polynucleotide or polypeptide sequence.
In another preferred embodiment of the invention there is provided a
computer readable medium having stored thereon a member selected from the
group consisting of: a polynucleotide comprising the sequence of SEQ ID
NO:1; a polypeptide comprising the sequence of SEQ ID NO:2; a set of
polynucleotide sequences wherein at least one of said sequences comprises
the sequence of SEQ ID NO:1; a set of polypeptide sequences wherein at
least one of said sequences comprises the sequence of SEQ ID NO:2; a data
set representing a polynucleotide sequence comprising the sequence of SEQ
ID NO:1; a data set representing a polynucleotide sequence encoding a
polypeptide sequence comprising the sequence of SEQ ID NO:2; a
polynucleotide comprising the sequence of SEQ ID NO:1; a polypeptide
comprising the sequence of SEQ ID NO:2; a set of polynucleotide sequences
wherein at least one of said sequences comprises the sequence of SEQ ID
NO:1; a set of polypeptide sequences wherein at least one of said
sequences comprises the sequence of SEQ ID NO:2; a data set representing a
polynucleotide sequence comprising the sequence of SEQ ID NO:1; a data set
representing a polynucleotide sequence encoding a polypeptide sequence
comprising the sequence of SEQ ID NO:2. A further preferred embodiment of
the invention provides a computer based method for performing homology
identification, said method comprising the steps of providing a
polynucleotide sequence comprising the sequences of SEQ ID NO:1 in a
computer readable medium; and comparing said polynucleotide sequence to at
least one polynucleotide or polypeptide sequence to identify homology.
A still further preferred embodiment of the invention provides a computer
based method for performing homology identification, said method
comprising the steps of: providing a polypeptide sequence comprising the
sequence of SEQ ID NO:2 in a computer readable medium; and comparing said
polypeptide sequence to at least one polynucleotide or polypeptide
sequence to identify homology.
A further embodiment of the invention provides a computer based method for
polynucleotide assembly, said method comprising the steps of: providing a
first polynucleotide sequence comprising the sequence of SEQ ID NO:1 in a
computer readable medium; and screening for at least one overlapping
region between said first polynucleotide sequence and a second
polynucleotide sequence.
A further embodiment of the invention provides a computer based method for
performing homology identification, said method comprising the steps of:
providing a polynucleotide sequence comprising the sequence of SEQ ID NO:1
in a computer readable medium; and comparing said polynucleotide sequence
to at least one polynucleotide or polypeptide sequence to identify
homology.
All publications and references, including but not limited to patents and
patent applications, cited in this specification are herein incorporated
by reference in their entirety as if each individual publication or
reference were specifically and individually indicated to be incorporated
by reference herein as being fully set forth. Any patent application to
which this application claims priority is also incorporated by reference
herein in its entirety in the manner described above for publications and
references.
GLOSSARY
The following definitions are provided to facilitate understanding of
certain terms used frequently herein.
"Antibody(ies)" as used herein includes polyclonal and monoclonal
antibodies, chimeric, single chain, and humanized antibodies, as well as
FAB fragments, including the products of an FAB or other immunoglobulin
expression library.
"Antigenically equivalent derivative(s)" as used herein encompasses a
polypeptide, polynucleotide, or the equivalent of either which will be
specifically recognized by certain antibodies which, when raised to the
protein, polypeptide or polynucleotide according to the invention,
interferes with the immediate physical interaction between pathogen and
mammalian host.
"Bispecific antibody(ies)" means an antibody comprising at least two
antigen binding domains, each domain directed against a different epitope.
"Bodily material(s) means any material derived from an individual or from
an organism infecting, infesting or inhabiting an individual, including
but not limited to, cells, tissues and waste, such as, bone, blood, serum,
cerebrospinal fluid, semen, saliva, muscle, cartilage, organ tissue, skin,
urine, stool or autopsy materials.
"Disease(s)" means any disease caused by or related to infection by a
bacteria.
"Fusion protein(s)" refers to protein encoded by two, often unrelated,
fused genes or fragments thereof. In one example, EP-A-0464 discloses
fusion proteins comprising various portions of constant region of
immunoglobulin molecules together with another human protein or part
thereof. In many cases, employing an immunoglobulin Fe region as a part of
a fusion protein is advantageous for use in therapy and diagnosis
resulting in, for example, improved pharmacokinetic properties [see, e.g.,
EP-A 0232262]. On the other hand, for some uses it would be desirable to
be able to delete the Fc part after the fusion protein has been expressed,
detected and purified.
"Host cell(s)" is a cell which has been transformed or transfected, or is
capable of transformation or transfection by an exogenous polynucleotide
sequence.
"Identify," as known in the art, is a relationship between two or more
polypeptide sequences or two or more polynucleotide sequences, as the case
may be, as determined by comparing the sequences. In the art, "identify"
also means the degree of sequence relatedness between polypeptide or
polynucleotide sequences, as the case may be, as determined by the match
between strings of such sequences. "Identity" can be readily calculated by
known methods, including but not limited to those described in
(Computational Molecular Biology, Lesk, A. M., ed., Oxford University
Press, New York, 1988; Biocomputing: Informatics and Genome Projects,
Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of
Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana
Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von
Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov,
M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo,
H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods to
determine identity are designed to give the largest match between the
sequences tested. Moreover, methods to determine identity are codified in
publicly available computer programs. Computer program methods to
determine identity between two sequences include, but are not limited to,
the GCG program package (Devereux, J., et al., Nucleic Acids Research
12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J.
Molec. Biol. 215: 403-410 (1990). The BLAST X program is publicly
available from NCBI and other sources (BLAST Manual, Atschul, S., et al.,
NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:
403-410 (1990). The well known Smith Waterman algorithm may also be used
to determine identity.
Parameters for polypeptide sequence comparison including the following:
Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)
Comparison matrix BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad.
Sci. USA. 89:10915-10919 (1992)
Gap Penalty: 12
Gap Length Penalty: 4
A program useful with these parameters is publicly available as the "gap"
program from Genetics Computer Group, Madison Wis. The aforementioned
parameters are the default parameters for peptide comparisons (along with
no penalty for end gaps).
Parameters for polynucleotide comparison include the following: Algorithm:
Needleman and Wunsch, J. Mol Biol. 48:443-453 (1970)
Comparison matrix: matches=+10, mismatch=0
Gap Penalty: 50
Gap Length Penalty: 3
Available as: The "gap" program from Genetics Computer Group, Madison, Wis.
These are the default parameters for nucleic acid comparisons.
A preferred meaning for "identity" for polynucleotides and polypeptides, as
the case may be, are provided in (1) and (2) below.
(1) Polynucleotide embodiments further include an isolated polynucleotide
comprising a polynucleotide sequence having at least a 50, 60, 70, 80, 85,
90, 95, 97 or 100% identity to the reference sequence of SEQ ID NO:1,
wherein said polynucleotide sequence may be identical to the reference
sequence of SEQ ID NO:1 or may include up to a certain integer number of
nucleotide alterations as compared to the reference sequence, wherein said
alterations are selected from the group consisting of at least one
nucleotide deletion, substitution, including transition and transversion,
or insertion, and wherein said alterations may occur at the 5' to 3'
terminal positions of the reference nucleotide sequence or anywhere
between those terminal positions, interspersed either individually among
the nucleotides in the reference sequence or in one or more contiguous
groups within the reference sequence, and wherein said number of
nucleotide alterations is determined by multiplying the total number of
nucleotides in SEQ ID NO:1 by the integer defining the percent identity
divided by 100 and then subtracting that product from said total number of
nucleotides in SEQ ID NO:1, or:
n.sub.n.ltoreq.x.sub.n -(x.sub.n.multidot.y),
wherein n.sub.n is the number of nucleotide alterations, x.sub.n is the
total number of nucleotides in SEQ ID NO:1, y is 0.50 for 50%, 0.60 for
60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%,
0.97 for 97% or 1.00 for 100%, and .multidot. is the symbol for the
multiplication operator, and wherein any non-integer product of x.sub.n
and y is rounded down to the nearest integer prior to subtracting it from
x.sub.n. Alterations of a polynucleotide sequence encoding the polypeptide
of SEQ ID NO:2 may create nonsense, missense or frameshift mutations in
this coding sequence and thereby alter the polypeptide encoded by the
polynucleotide following such alterations.
By way of example, a polynucleotide sequence of the present invention may
be identical to the reference sequence of SEQ ID NO:1, that is it may be
100% identical, or it may include up to a certain integer number of
nucleic acid alterations as compared to the reference sequence such that
the percent identity is less than 100% identity. Such alterations are
selected from the group consisting of at least one nucleic acid deletion,
substitution, including transition and transversion, or insertion, and
wherein said alterations may occur at the 5' or 3' terminal positions of
the reference polynucleotide sequence or anywhere between those terminal
positions, interspersed either individually among the nucleic acids in the
reference sequence or in one or more contiguous groups within the
reference sequence. The number of nucleic acid alterations for a given
percent identity is determined by multiplying the total number of nucleic
acids in SEQ ID NO:1 by the integer defining the percent identity by 100
and then subtracting that product from said total number of nucleic acids
in SEQ ID NO:1, or:
n.sub.n.ltoreq.x.sub.n -(x.sub.n.multidot.y),
wherein n.sub.n is the number of nucleic acid alterations, x.sub.n is the
total number of nucleic acids in SEQ ID NO:1, y is, for instance 0.70 for
70%, 0.80 for 80%, 0.85 for 85% etc., .multidot. is the symbol for the
multiplication operator, and wherein any non-integer product of x.sub.n
and y is rounded down to the nearest integer prior to subtracting from it
from x.sub.n.
(2) Polypeptide embodiments further include an isolated polypeptide
comprising a polypeptide having at least a 50, 60, 70, 80, 85, 90, 95, 97
or 100% identity to a polypeptide reference sequence of SEQ ID NO:2,
wherein said polypeptide sequence may be identical to the reference
sequence of SEQ ID NO:2 or may include up to a certain integer number of
amino acid alterations as compared to the reference sequence, wherein said
alterations are selected from the group consisting of at least one amino
acid deletion, substitution, including conservative and non-conservative
substitution, or insertion, and wherein said alterations may occur at the
amino- or carboxy-terminal positions of the reference polypeptide sequence
or anywhere between those terminal positions, interspersed either
individually among the amino acids in the reference sequence or in one or
more contiguous groups within the reference sequence, and wherein said
number of amino acid alterations is determined by multiplying the total
number of amino acids in SEQ ID NO:2 by the integer defining the percent
identity divided by 100 and then subtracting that product from said total
number of amino acids in SEQ ID NO:2, or:
n.sub.a.ltoreq.x.sub.a -(x.sub.a.multidot.y),
wherein n.sub.a is the number of amino acid alterations, x.sub.a is the
total number of amino acids in SEQ ID NO:2, y is 0.50 for 50%, 0.60 for
60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85% , 0.90 for 90%. 0.95 for
95%. 0.97 for 97% or 1.00 for 100%, and .multidot. is the symbol for the
multiplication operator, and wherein any non-integer product of x.sub.a
and y is rounded down to the nearest integer prior to subtracting it from
x.sub.a.
By way of example, a polypeptide sequence of the present invention may be
identical to the reference sequence of SEQ ID NO:2, that is it may be 100%
identical, or it may include up to a certain integer number of amino acid
alterations as compared to the reference sequence such that the percent
identity is less than 100% identity. Such alterations are selected from
the group consisting of at least one amino acid deletion, substitution,
including conservative and non-conservative substitution, or insertion,
and wherein said alterations may occur at the amino- or carboxy-terminal
positions of the reference polypeptide sequence or anywhere between those
terminal positions, interspersed either individually among the amino acids
in the reference sequence or in one or more contiguous groups within the
reference sequence. The number of amino acid alterations for a given %
identity is determined by multiplying the total number of amino acids in
SEQ ID NO:2 by the integer defining the percent identity divided by 100
and then subtracting that product from said total number of amino acids in
SEQ ID NO:2, or:
n.sub.a.ltoreq.x.sub.a -(x.sub.a 19 y),
wherein n.sub.a is the number of amino acid alterations, x.sub.a is the
total number of amino acids in SEQ ID NO:2, y is, for instance 0.70 for
70%, 0.80 for 80%, 0.85% etc., and .multidot. is the symbol for the
multiplication operator, and wherein the non-integer product of x.sub.a
and y is rounded down to the nearest integer prior to subtracting it from
x.sub.a.
"Immunologically equivalent derivative(s)" as used herein encompasses a
polypeptide, polynucleotide, or the equivalent of either which when used
in a suitable formulation to raise antibodies in a vertebrate, the
antibodies act to interfere with the immediate physical interaction
between pathogen and mammalian host.
"Immunospecific" means that characterstic of an antibody whereby it
possesses substantially greater affinity for the polypeptides of the
invention or the polynucleotide of the invention than its affinity for
other related polypeptides or polynucleotides respectively, particularly
those polypeptides and polynucleotides in the prior art.
"Individual(s)" means a multicellular eukaryote, including, but not limited
to metazoan, a mammal, an ovid, a bovid, a simian, a primate, and a human.
"Isolated" means altered"by the hand of man" from its natural state, i.e.,
if it occurs in nature, it has been changed or removed from its original
environment, or both. For example, a polynucleotide or a polypeptide
naturally present in a living organism is not "isolated," but the same
polynucleotide or polypeptide separated from the coexisting materials of
its natural state is "isolated", as the term is employed herein. Moreover,
a polynucleotide or polypeptide that is introduced into an organism by
transformation, genetic manipulation or by an other recombinant method is
"isolated" even if it is still present in said organism, which organism
may be living or non-living.
"Organism(s)" means a (i) prokaryote, including but not limited to, a
member of the genus Streptococcus, Staphylococcus, Bordetella,
Corynebacterium, Mycobacterium, Neisseria, Haemophilus, Actinomycetes,
Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella, Pasturella,
Moraxella, Acinetobacter, Erysipelothrix, Branhamella, Actinobacillus,
Streptobacillus, Listeria, Calymmatobacterium, Brucella, Bacillus,
Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella, Vibrio,
Proteus, Erwina, Borrelia, Leptospira, Spirillum, Campylobacter, Shigella,
Legionella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borrelia and
Mycoplasma, and further including, but not limited to, a member of the
species or group, Group A Streptococcus, Group B Streptococcus, Group C
Streptococcus, Group D Streptococcus, Group G Streptococcus, Streptococcus
pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae,
Streptococcus faecalis, Streptococcus faecium, Streptococcus durans,
Neisseria gonorrheae, Neisseria meningitidis, Staphylococcus aureus,
Staphylococcus epidermidis, Corynebacterium diptheriae, Gardnerella
vaginalis, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium
ulcerans, Mycobacterium leprae, Actinomyctes israelii, Listeria
monocytogenes, Bordetella pertusis, Bordatella parapertusis, Bordetella
bronchiseptica, Escherichia coli, Shigella dysenteria, Haemophilus
influenzae, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus
ducreyi, Bordetella, Salmonella typhi, Citrobacter freundii, Proteus
mirabilis, Proteus vulgaris, Yersinia pestis, Kleibsiella pneumoniae,
Serratia marcessens, Serratia liquefaciens, Vibrio cholera, Shigella
dysenterii, Shigella flexneri, Pseudomonas aeruginosa, Franscisella
tularensis, Brucella abortis, Bacillus anthracts, Bacillus cereus,
Clostridium perfringens, Clostridium tetani, Clostridium botulinum,
Treponema pallidum, Rickettsia rickettsii and Chlamydia trachomitis, (ii)
in archaeon, including but not limited to Archaebacter, and (iii) a
unicellular or filamentous eukaryote, including but not limited to, a
protozoan, a fungus, a member of the genus Saccharomyces, Kluveromyces, or
Candida, and a member of the species Saccharomyces ceriviseae,
Kluveromyces lactis, or Candida albicans.
"Polynucleotide(s)" generally refers to any polyribonucleotide or
polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified
RNA or DNA. "Polynucleotide(s)" include, without limitation single- and
double-stranded DNA, DNA that is a mixture of single- and double-stranded
regions or single-, double- and triple-stranded regions, single- and
double-stranded RNA, and RNA that is mixture of single- and
double-stranded regions, hybrid molecules comprising DNA and RNA that may
be single-stranded or, more typically, double-stranded, or triple-stranded
regions, or a mixture of single- and double-stranded regions. In addition,
"polynucleotide" as used herein refers to triple-stranded regions
comprising RNA or DNA or both RNA and DNA. The strands in such regions may
be from the same molecule or from different molecules. The regions may
include all of one or more of the molecules, but more typically involve
only a region of some of the molecules. One of the molecules of a
triple-helical region often is an oligonucleotide. As used herein, the
term "polynucleotide(s)" also includes DNAs or RNAs as described above
that contain one or more modified bases. Thus, DNAs or RNAs with backbones
modified for stability or for other reasons are "polynucleotide(s)" as
that term is intended herein. Moreover, DNAs or RNAs comprising unusual
bases, such as inosine, or modified bases, such as tritylated bases, to
name just two examples, are polynucleotides as the term is used herein. It
will be appreciated that a great variety of modifications have been made
to DNA and RNA that serve many useful purposes known to those of skill in
the art. The term "polynucleotide(s)" as it is employed herein embrances
such chemically, enzymatically or metabolically modified forms of
polynucleotides, as well as the chemical forms of DNA and RNA
characteristics of viruses and cells, including, for example, simple and
complex cells. "Polynucleotide(s)" also embrances short polynucleotides
often referred to as oligonucleotide(s).
"Polypeptide(s)" refers to any peptide or protein comprising two or more
amino acids joined to each other by peptide bonds or modified peptide
bonds. "Polypeptide(s)" refers to both short chains, commonly referred to
as peptides, oligopeptides and oligomers and to longer chains generally
referred to as proteins. Polypeptides may contain amino acids other than
the 20 gene encoded amino acids. "Polypeptide(s)" include those modified
either by natural processes, such as processing and other
post-translational modifications, but also by chemical modification
techniques. Such modifications are well described in basic texts and in
more detailed monographs, as well as in a voluminous research literature,
and they are well known to those of skill in the art. It will be
appreciated that the same type of modification may be present in the same
or varying degree at several sites in a given polypeptide. Also, a given
polypeptide may contain many types of modifications. Modifications can
occur anywhere in a polypeptide, including the peptide backbone, the amino
acid side-chains, and the amino or carboxyl termini. Modifications
include, for example, acetylation, acylation, ADP-ribosylation, amidation,
covalent attachment of flavin, covalent attachment of a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative, covalent
attachment of a lipid or lipid derivative, covalent attachment of
phosphotidylinositol, cross-linking, cyclization, disulfide bond
formation, demethylation, formation of covalent cross-links, formation of
cysteine, formation of pyroglutamate, formylation, gamma-carboxylation,
GPI anchor formation, hydroxylation, iodination, methylation,
myristoylation, oxidation, proteolytic processing, phosphorylation,
prenylation, racemization, glycosylation, lipid attachment, sulfation,
gamma-carboxylation of glutamic acid residues, hydroxylation and
ADP-ribosylation, selenoylation, sulfation, transfer-RNA mediated addition
of amino acids to proteins, such as arginylation, and ubiquitination. See,
for instance, PROTEINS--STRUCTURE AND MOLECULAR PROPERTIES, 2nd, Ed., T.
E. Creighton, W. H. Freeman and Company, New York (1993) and Wold, F.,
Posttranslational Protein Modifications: Perspective and Prospects pgs.
1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.
Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth.
Enzymol. 182:626-646 (1990) and Rattan et al., Protein Synthesis:
Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663: 48-62
(1992). Polypeptide may be branded or cyclic, with or without branching.
Cyclic, branched and branched circular polypeptides may result from
post-translational natural processes and may be made by entirely synthetic
methods as well.
"Recombinant expression system(s)" refers to expression systems or portions
thereof or polynucleotides of the invention introduced or transformed into
a host cell or host cell lysate for the production of the polynucleotides
and polypeptides of the invention.
"Subtraction set" is one or more, but preferably less than 100,
polynucleotides comprising at least one polynucleotide of the invention.
"Variant(s)" as the term is used herein, is a polynucleotide or polypeptide
that differs from a reference polynucleotide or polypeptide respectively,
but retains essential properties. A typical variant of a polynucleotide
differs in nucleotide sequence from another, reference polynucleotide.
Changes in the nucleotide sequence of the variant may or may not alter the
amino acid sequence of a polypeptide encoded by the reference
polynucleotide. Nucleotide changes may result in amino acid substitutions,
additions, deletions, fusion proteins and truncations in the polypeptide
encoded by the reference sequence, as discussed below. A typical variant
of a polypeptide differs in amino acid sequence from another, reference
polypeptide. Generally, differences are limited so that the sequences of
the reference and the variant are closely similar overall and, in many
regions, identical. A variant and reference polypeptide may differ in
amino acid sequence by one or more substitutions, additions, deletions in
any combination. A substituted or inserted amino acid residue may or may
not be one encoded by the genetic code. The present invention also
includes include variants of each of the polypeptides of the invention,
that is polypeptides that vary from the referents by conservative amino
acid substitutions, whereby a residue is substituted by another with like
characteristics. Typical such substitutions are among Ala, Val, Leu and
Ile; among Ser and Thr; among the acidic residues Asp and Glu; among Asn
and Gln; and among the basic residues Lys and Arg; or aromatic residues
Phe and Tyr. Particularly preferred are variants in which several, 5-10,
1-5, 1-3, 1-2 or 1 amino acids are substituted, deleted, or added in any
combination. A variant of a polynucleotide or polypeptide may be a
naturally occurring such as an allelic variant, or it may be a variant
that is not known to occur naturally. Non-naturally occurring variants of
polynucleotides and polypeptides may be made by mutagenesis techniques, by
direct synthesis, and by other recombinant methods known to skilled
artisans.
EXAMPLES
The examples below are carried out using standard techniques, which are
well known and routine to those of skill in the art, except where
otherwise described in detail. The examples are illustrative, but do not
limit the invention.
Example 1
Strain selection, Library Production and Sequencing
The polynucleotide having a DNA sequence given in Table 1 [SEQ ID NO:1] was
obtained from a library of clones of chromosomal DNA of Pseudomonas
aeruginosa in E. coli. The sequencing data from two or more clones
containing overlapping Pseudomonas aeruginosa DNAs was used to construct
the contiguous DNA sequence in SEQ ID NO:1. Libraries may be prepared by
routine methods, for example:
Methods 1 and 2 below.
Total cellular DNA is isolated from Pseudomonas aeruginosa Pseudomonas
aeruginosa strain 4 according to standard procedures and size-fractionated
by either of two methods.
Method 1
Total cellular DNA is mechanically sheared by passage through a needle in
order to size-fractionate according to standard procedures. DNA fragments
of up to 11 kbp in size are rendered blunt by treatment with exonuclease
and DNA polymerase, and EcoRI linkers added. Fragments are ligated into
the vector Lambda ZapII that has been cut with EcoRI, the library packaged
by standard procedures and E. coli infected with the packaged library. The
library is amplified by standard procedures.
Method 2
Total cellular DNA is partially hydrolized with a one or a combination of
restriction enzymes appropriate to generate a series of fragments for
cloning into library vectors (e.g., RsaI, PalI, AlI, Bshl235I), and such
fragments and size-fractioned according to standard procedures EcoRI
linkers are ligated to the DNA and the fragments then ligated into the
vector Lambda ZapII that have been cut with EcoRI, the library packaged by
standard procedures, and E. coli infected with the packaged library. The
library is amplified by standard procedures.
Example 2
ups (ugc) Characterization
We have used chemical mutagenesis to isolate Ts mutants in an attempt to
identify essential P. aeruginosa gene products. Over 100 mutants, which
show Ts growth on complex medium at 44.degree. C., have been isolated. A
genomic library containing 5 to 6 kb DNA fragments of P. aeruginosa was
constructed to complement these Ts mutants. Nucleotide sequence analysis
of plasmids complementing the Ts mutant revealed many known essential
genes as well as genes with unknown functions. One of the novel essential
genes, ugc (upstream gene of cdsA), encodes a polypeptide that has 40%
identical to the undecaprenyl diphosphate synthase (Ups) from Micrococcus
luteus and therefore ugc is renamed as ups. Nucleotide sequence analysis
of the Ts ups allele revealed a C.fwdarw.T transition mutation at
nucleotide position 404 in Table 1 [SEQ ID NO:1], which caused an amino
acid substitution resulting in the change of alanine at position 135 in
Table 1 [SEQ ID NO:2] to valine in the Ups ORF. Flow cytometry studies of
the ups Ts mutant revealed that, following a shift to the non-permissive
temperature, both cellular membrane potential and integrity were disrupted
and cell lysis rapidly followed. Viable cell count data supported this
rapid loss of viability. In contrast, there were no obvious effects on the
complemented mutant at the non-permissive temperature. Therefore, these
results demonstrate that the ups gene is essential for cell viability and
could be used as an antimicrobial target.
SEQUENCE LISTING
<100> GENERAL INFORMATION:
<160> NUMBER OF SEQ ID NOS: 2
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 1
<211> LENGTH: 756
<212> TYPE: DNA
<213> ORGANISM: Pseudomonas aeruginosa
<220> FEATURE:
<221> NAME/KEY: CDS
<222> LOCATION: (1)...(753)
<400> SEQUENCE: 1
atg gaa aag acc cgg aag gat gtg tgc gtg cca cgc cac gtg gcc att 48
Met Glu Lys Thr Arg Lys Asp Val Cys Val Pro Arg His Val Ala Ile
1 5 10 15
atc atg gac ggt aac aat cgc tgg gcg aag aag cgt ctt ctg ccc ggc 96
Ile Met Asp Gly Asn Asn Arg Trp Ala Lys Lys Arg Leu Leu Pro Gly
20 25 30
gtc gcc ggc cac aag gcc ggt gtc gat gcc gtc agg gcg gtg atc gag 144
Val Ala Gly His Lys Ala Gly Val Asp Ala Val Arg Ala Val Ile Glu
35 40 45
gtc tgc gcc gag gca ggg gtc gag gtc ctc acc ctg ttc gcg ttc tcc 192
Val Cys Ala Glu Ala Gly Val Glu Val Leu Thr Leu Phe Ala Phe Ser
50 55 60
agc gag aac tgg cag cgt ccg gcg gac gaa gtc agc gcg ctg atg gag 240
Ser Glu Asn Trp Gln Arg Pro Ala Asp Glu Val Ser Ala Leu Met Glu
65 70 75 80
ctg ttt ctc gtg gcc ctg cgc cgc gag gtg cgc aag ctc gac gag aac 288
Leu Phe Leu Val Ala Leu Arg Arg Glu Val Arg Lys Leu Asp Glu Asn
85 90 95
ggc atc cgc ctg cgc atc atc ggc gat cgc acg cgt ttc cat ccg gag 336
Gly Ile Arg Leu Arg Ile Ile Gly Asp Arg Thr Arg Phe His Pro Glu
100 105 110
ttg cag gcg gcc atg cgc gaa gcg gaa gcc gcc act gcc ggc aat acc 384
Leu Gln Ala Ala Met Arg Glu Ala Glu Ala Ala Thr Ala Gly Asn Thr
115 120 125
cgt ttc ctc ctc cag gtc gcc gcc aac tac ggc ggc cag tgg gac atc 432
Arg Phe Leu Leu Gln Val Ala Ala Asn Tyr Gly Gly Gln Trp Asp Ile
130 135 140
gtc cag gcc gca cag cgc ctg gcg cgc gag gtc cag ggc ggg cac ctg 480
Val Gln Ala Ala Gln Arg Leu Ala Arg Glu Val Gln Gly Gly His Leu
145 150 155 160
gcg gcg gac gat atc tcc gcc gag ctg ctc cag ggc tgc ctg gtg acc 528
Ala Ala Asp Asp Ile Ser Ala Glu Leu Leu Gln Gly Cys Leu Val Thr
165 170 175
ggc gac cag ccg ctg ccc gac ctg tgc atc cgc acc ggc ggc gag cat 576
Gly Asp Gln Pro Leu Pro Asp Leu Cys Ile Arg Thr Gly Gly Glu His
180 185 190
cgc atc agc aat ttc ctt ctc tgg cag ctg gcc tac gcc gag ctg tat 624
Arg Ile Ser Asn Phe Leu Leu Trp Gln Leu Ala Tyr Ala Glu Leu Tyr
195 200 205
ttc tcc gac ctg ttc tgg ccc gac ttc aag cac gcg gcg atg cgg gct 672
Phe Ser Asp Leu Phe Trp Pro Asp Phe Lys His Ala Ala Met Arg Ala
210 215 220
gcc ctg gcg gat ttc tcc aag cgc cag cgc cgc ttc ggc aag acc agc 720
Ala Leu Ala Asp Phe Ser Lys Arg Gln Arg Arg Phe Gly Lys Thr Ser
225 230 235 240
gag caa gtc gag gcc gaa gcc cgt ccg tca tgc tga 756
Glu Gln Val Glu Ala Glu Ala Arg Pro Ser Cys
245 250
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 2
<211> LENGTH: 251
<212> TYPE: PRT
<213> ORGANISM: Pseudomonas aeruginosa
<400> SEQUENCE: 2
Met Glu Lys Thr Arg Lys Asp Val Cys Val Pro Arg His Val Ala Ile
1 5 10 15
Ile Met Asp Gly Asn Asn Arg Trp Ala Lys Lys Arg Leu Leu Pro Gly
20 25 30
Val Ala Gly His Lys Ala Gly Val Asp Ala Val Arg Ala Val Ile Glu
35 40 45
Val Cys Ala Glu Ala Gly Val Glu Val Leu Thr Leu Phe Ala Phe Ser
50 55 60
Ser Glu Asn Trp Gln Arg Pro Ala Asp Glu Val Ser Ala Leu Met Glu
65 70 75 80
Leu Phe Leu Val Ala Leu Arg Arg Glu Val Arg Lys Leu Asp Glu Asn
85 90 95
Gly Ile Arg Leu Arg Ile Ile Gly Asp Arg Thr Arg Phe His Pro Glu
100 105 110
Leu Gln Ala Ala Met Arg Glu Ala Glu Ala Ala Thr Ala Gly Asn Thr
115 120 125
Arg Phe Leu Leu Gln Val Ala Ala Asn Tyr Gly Gly Gln Trp Asp Ile
130 135 140
Val Gln Ala Ala Gln Arg Leu Ala Arg Glu Val Gln Gly Gly His Leu
145 150 155 160
Ala Ala Asp Asp Ile Ser Ala Glu Leu Leu Gln Gly Cys Leu Val Thr
165 170 175
Gly Asp Gln Pro Leu Pro Asp Leu Cys Ile Arg Thr Gly Gly Glu His
180 185 190
Arg Ile Ser Asn Phe Leu Leu Trp Gln Leu Ala Tyr Ala Glu Leu Tyr
195 200 205
Phe Ser Asp Leu Phe Trp Pro Asp Phe Lys His Ala Ala Met Arg Ala
210 215 220
Ala Leu Ala Asp Phe Ser Lys Arg Gln Arg Arg Phe Gly Lys Thr Ser
225 230 235 240
Glu Gln Val Glu Ala Glu Ala Arg Pro Ser Cys
245 250
Top